Methods for rna sequencing

ABSTRACT

This invention provides methods for detecting RNAs in a biological sample while using only a small amount of sample input, e.g., less than or equal to 1 mL. The methods described herein are able to detect a large percentage of protein-coding genes having the ENSEMBL gene annotation HG38.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT/US2018/032959, filed May 16,2018, which application claims priority to U.S. Provisional ApplicationNo. 62/553,691, filed Sep. 1, 2017, the disclosures of which are herebyincorporated by reference in their entireties for all purposes.

FIELD OF THE INVENTION

The invention relates to the field of nucleic acid sequencing andlibrary construction.

BACKGROUND

While nucleic acid molecules encode valuable information about asubject's genetic makeup and disease conditions, sequencing nucleic acidmolecules, especially ribonucleic acids (RNAs), has been challenging.Traditional RNA sequencing methods often require a large amount ofbiological sample to obtain enough RNAs for downstream sequencing and/orlibrary construction. The isolation and purification of RNAs from thesample is a complicated and time consuming process that often requiresthe use of multiple reagents, prolonged periods of protein precipitationand washing, and sometimes exposure to high temperature and high saltsolutions. In addition to the multistep process, the RNAs may undergodegradation or unwanted modification, making downstream sequencingreactions less accurate. There exists a need for novel and efficientmethods to sequencing RNAs from biological samples.

SUMMARY OF THE INVENTION

This invention provides methods for detecting RNAs in a biologicalsample (e.g., a cell-free biological sample) while using only a smallamount of sample input, e.g., less than or equal to 1 mL. Depending onthe biological sample, in some embodiments, the methods described hereinmay detect at least about 100 different genes. As demonstrated inExamples 1-3, the methods detected a large percentage of protein-codinggenes having the ENSEMBL gene annotation HG38.

In one aspect, the invention provides a method for detecting a pluralityof ribonucleic acids (RNAs) in a biological sample from a subject by (a)constructing a cDNA library from the plurality of RNAs in the biologicalsample, wherein an effective volume of the biological sample used toconstruct the cDNA library is less than or equal to about 1 mL; and (b)detecting RNA genome equivalents in the cDNA library.

In some embodiments, the effective volume of the biological sample isless than or equal to about 500 μL (e.g., less than or equal to about250 μL, less than or equal to about 100 μL, less than or equal to about50 μL, less than or equal to about 25 μL, or less than or equal to about10 μL). In other embodiments, the effective volume of the biologicalsample is between about 1 μL and about 500 μL (e.g., between about 1 μLand about 250 μL, between about 1 μL and about 100 μL, between about 1μL and about 50 μL, between about 1 μL and about 25 μL, between about 1μL and about 10 μL, between about 5 μL and about 100 μL, between about 5μL and about 50 μL, between about 5 μL and about 25 μL, between about 5μL and about 10 μL, between about 10 μL and about 100 μL, between about10 μL and about 50 μL, between about 10 μL and about 25 μL, betweenabout 25 μL and about 100 μL, between about 25 μL and about 50 μL, orbetween about 50 μL and about 100 μL).

In some embodiments, the biological sample is a cell-free biologicalsample.

In some embodiments, at least about 100 different genes (e.g., at leastabout 1,000 different genes, at least about 5,000 different genes, atleast about 10,000 different genes, at least about 20,000 differentgenes, at least about 30,000 different genes, at least about 40,000different genes, or at least about 50,000 different genes) are detected.In some embodiments, the different genes comprise different categoriesof RNAs (e.g., mRNA, lincRNA, miRNA, snRNA, and combinations thereof).

In some embodiments, the biological sample is a whole blood sample, aplasma sample, a serum sample, a saliva sample, a cell culture mediasample, a urine sample, an amniotic fluid sample, a mucus sample, asemen sample, a vaginal fluid sample, a sputum sample, a cerebrospinalfluid sample, a lymphatic fluid sample, an ocular fluid sample, a sweatsample, or a stool sample. In particular embodiments, the biologicalsample is a serum sample, a plasma sample, a saliva sample, or a cellculture media sample. In particular embodiments, the cell culture mediasample is a serum sample or a plasma sample. In particular embodiments,the cell culture media sample is an in vitro fertilization (IVF) culturemedia sample.

In some embodiments, step (a) of the method comprises: (a1) breaking uplipid bilayers and/or ribonucleoprotein complexes in the biologicalsample; (a2) removing deoxyribonucleic acids (DNAs) in the biologicalsample; (a3) synthesizing a plurality of short, double-strandedcomplementary DNAs (cDNAs) oligonucleotides using the RNAs in thebiological sample as templates; (a4) ligating at least one adaptor to anend of each short, double-stranded cDNA oligonucleotide to generate aplurality of adaptor-ligated, double-stranded cDNA oligonucleotides;(a5) amplifying the plurality of adaptor-ligated, double-stranded cDNAoligonucleotides using primers that hybridize to the adaptor; (a6)selecting one strand of each adaptor-ligated, double-stranded cDNAoligonucleotide; and (a7) amplifying the selected strand of eachadaptor-ligated, double-stranded cDNA oligonucleotide to generate thecDNA library, wherein the cDNA library comprises cDNA oligonucleotideshaving the same sequences as the sequences of the genomic DNAs fromwhich the RNAs are transcribed.

In some embodiments, step (a4) described above comprises ligating twoadaptors each to one end of each short, double-stranded cDNAoligonucleotide to generate a plurality of adaptor-ligated,double-stranded cDNA oligonucleotides. In particular embodiments, thetwo adaptors are the same. In particular embodiments, the two adaptorsare different. In some embodiments, one of the adaptors comprises adegradable sequence. In particular embodiments, the degradable sequencecomprises a uracil DNA glycosylase recognition sequence.

In some embodiments, step (a3) described above comprises: (a3.1)synthesizing a plurality of first strand cDNAs by reverse transcribingthe RNAs in the biological sample; (a3.2) fragmenting the plurality offirst strand cDNAs to generate a plurality of cDNA fragments; (a3.3)ligating a 3′ primer to the 3′ end of each cDNA fragment in theplurality of cDNA fragments; and (a3.4) synthesizing the plurality ofshort, double-stranded cDNA oligonucleotides using a targeting primerand the plurality of cDNA fragments as templates, wherein the targetingprimer comprises a first portion comprising a sequence complementary tothe sequence of the 3′ primer and a second portion comprising adegradable sequence. In particular embodiments, the degradable sequencecomprises a uracil DNA glycosylase recognition sequence. In particularembodiments, the 3′ primer is an oligonucleotide comprising identicalnucleotides (e.g., a poly(G) oligonucleotide, a poly(C) oligonucleotide,a poly(A) oligonucleotide, a poly(T) oligonucleotide, or a poly(U)oligonucleotide). In particular embodiments, the 3′ primer is a poly(G)oligonucleotide. In particular embodiments, the sequence of the firstportion is a poly(C) oligonucleotide.

In some embodiments, the method further comprises removing the RNAs inthe biological sample. The RNAs may be removed using a metal ion and/orheat shock.

In some embodiments of the method, the plurality of adaptor-ligated,double-stranded cDNA oligonucleotides is purified using a charge-basedpurification method prior to step (a5) described above. In particularembodiments, the charge-based purification method comprises using beads.

In particular embodiments, in step (a6) described above, the strandcomprising the adaptor that comprises the degradable sequence isdegraded. In some embodiments, the strand is degraded by a uracil DNAglycosylase.

In some embodiments of the method, the method further comprises removingcDNA oligonucleotides that encode ribosomal RNAs (rRNAs) after step (a6)and prior to step (a7). In particular embodiments, an rRNA primer isused to target the cDNA oligonucleotides encoding rRNAs.

In some embodiments of the method, step (b) comprises: (bl) sequencingthe cDNA library to detect the RNA genome equivalents. The methodsdescribed herein may generate any number of sequencing reads. Inparticular embodiments, at least about 1,000 sequencing reads (e.g., atleast about 5,000 sequencing reads, at least about 10,000 sequencingreads, at least about 50,000 sequencing reads, at least about 100,000sequencing reads, at least about 500,000 sequencing reads, at leastabout 1 million sequencing reads, at least about 1.5 million sequencingreads, at least about 2 million sequencing reads, at least about 2.5million sequencing reads, at least about 5 million sequencing reads, atleast about 10 million sequencing reads, at least about 25 millionsequencing reads, or at least about 50 million sequencing reads) areobtained. In some embodiments, step (b) further comprises: (b2) mappingthe RNA genome equivalents detected to different categories of RNAs.

Other inventive products, methods, and features that can be used aloneor in combination with the aforesaid methods are evidenced by thedescription and examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exemplary schematic flowchart showing non-limiting steps inthe method of the invention.

FIGS. 2A-2I are bar graphs showing the distribution of the TPM(transcripts per million) values of genes specific to each of the threetissues in serum A (FIGS. 2A-2C), serum B (FIGS. 2D-2F), and serum C(FIGS. 2G-2I).

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The invention features a technology that is able to construct sequencinglibraries in large scale with small amounts of biological sample such asunprocessed serum as the direct input. Using a small amount ofbiological sample (e.g., a cell-free biological sample) such asunprocessed serum, the technology may allow the detection of at least100 different genes. For example, as demonstrated in Examples 1-3, withabout 60 μL of unprocessed serum, we detected over 50,000 out of all60,675 human genes among 42 different gene categories (ENSEMBL geneannotation (HG38)). Further, we were also able to detect tissue-specificgenes in serum. The technology could be applied to different liquidbiopsy types, such as saliva and in vitro fertilization (IVF) culturemedia, for the diagnosis and prognosis of various diseases.

II. Definitions

As used herein, the term “biological sample” refers to a biologicalsample obtained from a subject that can either be directly used in themethods of the invention or be processed before being used in themethods of the invention. Examples of a biological sample from a subjectinclude, but are not limited to, a whole blood sample, a plasma sample,a serum sample, a saliva sample, a cell culture media sample, a urinesample, an amniotic fluid sample, a mucus sample, a semen sample, avaginal fluid sample, a sputum sample, a cerebrospinal fluid sample, alymphatic fluid sample, an ocular fluid sample, a sweat sample, and astool sample. In some embodiments, a biological sample may be acell-free biological sample, which refers to a biological sampleobtained from a subject (e.g., a human) that does not contain any cellsor is subsequently processed to remove substantially all of the cellspresent in the sample. For example, a whole blood sample from a subjectmay be filtered to remove cells, e.g., red blood cells, white bloodcells, and platelets. In some embodiments, a cell-free biological samplemay also be constructed from a biological material obtained from asubject. For example, during IVF, a mature egg may be collected from thesubject and fertilized by sperm in a laboratory. The cell culture mediaused to culture the fertilized egg is an example of a cell-freebiological sample.

As used herein, the term “effective volume” refers to the volume of thebiological sample (e.g., a cell-free biological sample) that may allowthe detection of at least about 100 different genes in the biologicalsample. For example, if 5 biological samples each containing 10 μL areused to construct 5 cDNA libraries and the sequencing reads from eachcDNA library are about 6 million for a total of about 30 millionsequencing reads (6 million×5 samples), then the effective volume of thebiological sample is 50 μL (10 μL×5 samples). In another example, if one50 μL biological sample is sequenced 5 times, in which the sequencingreads each time are about 6 million for a total of 30 million sequencingreads after 5 times, then the effective volume of the biological sampleis 50 μL.

As used herein, the term “RNA genome equivalent” refers to the sequenceof the RNA that is amplified in the cDNA library constructed based onthe RNAs present in the biological sample (e.g., a cell-free biologicalsample). The cDNA library is then sequenced to detect the RNA genomeequivalents, thus, detecting the RNAs present in the original biologicalsample.

As used herein, the term “genomic DNA” refers to the DNA from which theRNAs are transcribed. The term “genomic DNA” does not refer to DNAs thatmay present in the biological sample (e.g., a cell-free biologicalsample). Genomic DNA is DNA that constitutes the genome of the organism.

As used herein, the term “cDNA” or “complementary DNA” refers to a DNAoligonucleotide synthesized from an RNA template either directly or insubsequent amplifications. A cDNA oligonucleotide may be generated usingan RNA as the template in a reaction catalyzed by the enzyme reversetranscriptase. This cDNA oligonucleotide may further be used as thetemplate to synthesis other cDNA oligonucleotides, e.g., a second cDNAoligonucleotide generated based on a first cDNA oligonucleotide as thetemplate. As described in detail herein, double-stranded cDNAoligonucleotides containing a first strand cDNA oligonucleotide(generated based on the RNAs in the biological sample (e.g., a cell-freebiological sample) as the template) and a second cDNA oligonucleotide(generated based on the first strand cDNA oligonucleotide as thetemplate) are made prior to adaptor ligation. Eventually, the secondstrand cDNA oligonucleotides are targeted for degradation and the firststrand cDNA oligonucleotides are selected for the second stage of PCRamplification to construct the cDNA library. Note that the sequences ofthe first cDNA oligonucleotides are the same as the sequences of thegenomic DNAs that are used to generate the RNAs in the biologicalsample. Note that the sequences of the second cDNA oligonucleotides arethe same as the sequences of the RNAs, except that the uracils arereplaced with thymidines.

As used herein, the term “gene” refers to a component of an organism'sgenome that is made of deoxyribonucleic acids (DNAs) and/or ribonucleicacids (RNAs). A gene may be a coding gene, which encodes the geneticsequence of a protein or peptide that is eventually expressed after genetranscription and translation. A gene may be a non-coding gene, whichencodes a genetic sequence that may not be expressed into a protein orpeptide and serves other functions in the organism's genome, such asregulating the expression of other genes.

As used herein, the term “lipid bilayers” refers to any cell membranesor vesicle membranes. Cell membranes or vesicle membranes may enclose acell, enclose a vesicle, or present inside a cell or a vesicle. Forexample, lipid bilayers may be the outer membrane of a cell. In otherembodiments, lipid bilayers may be the membrane of a vesicle, e.g., anexosome.

As used herein, the term “adaptor” refers to short, double-strandedoligonucleotides that are ligated to the ends of double-stranded cDNAoligonucleotides before the first stage of PCR amplification. Theprimers used during the first stage of PCR amplification may be designedto anneal to the adaptors in a manner such that only one type ofligation product is amplified. As described in detail further herein, aforward adaptor may include a degradable sequence (e.g., a uracil DNAglycosylase recognition sequence (e.g., a sequence containing dUTP)),which can be recognized by a uracil DNA glycosylase during downstreamstrand selection and degradation.

As used herein, the term “degradable sequence” refers to any nucleotidesequence that can be recognized by a protein, which then targets theoligonucleotide containing the degradable sequence for degradation. Forexample, the enzyme uracil DNA glycosylase recognizes a sequencecontaining dUTP (e.g., a uracil DNA glycosylase recognition sequence)and targets the oligonucleotide containing such a sequence fordegradation.

As used herein, the term “3′ primer” refers to a short oligonucleotidethat is ligated to the 3′ end of each cDNA fragment before the synthesisof the second strand cDNA oligonucleotides. In some embodiments, a 3′primer comprises identical nucleotides (e.g., a poly(G) oligonucleotide,a poly(C) oligonucleotide, a poly(A) oligonucleotide, a poly(T)oligonucleotide, or a poly(U) oligonucleotide).

As used herein, the term “charge based nucleotide purification method”refers to a method for nucleotide purification that relies on theinteractions between the oligonucleotide and the solid phase based ontheir charges. In some embodiments, a charge based nucleotidepurification method may use silica based columns or silica coated beads(e.g., silica coated magnetic beads).

As used herein, the term “about” refers to a range of values that is±20% of the specific value. For example, “about 30 million” includes±20% of 30 million, or from 24 million to 36 million. When the specificvalue is a percentage, the upper limit is 100%. Thus, about 95% refersto from 75% to 100%. Such a range performs the desired function orachieves the desired result. For example, “about” may refer to an amountthat is within less than 20% of, less than 10% of, within less than 5%of, within less than 1% of, within less than 0.1% of, and within lessthan 0.01% of the specific value.

As used herein, the term “between” refers to any quantity within therange indicated and enclosing each of the ends of the range indicated.For example, between 50 μL and 100 μL refers to any quantity within 50μL and 100 μL, as well as 50 μL and 100 μL. The term “between about,”e.g., between about 50 μL and about 100 μL, refers to any quantitywithin the range indicated and enclosing 50 μL−(50 μL×20%) as the lowerbound and 100 μL+(100 μL×20%) as the upper bound. For example, “betweenabout 50 μL and about 100 μL” refers to from 40 μL to 120 μL.

As used herein, the term “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to, mice,murines, rats, simians, humans, farm animals, sport animals, and pets.

III. Membrane Lysis and RNA Isolation

In certain aspects, the methods of the invention include a first step ofbreaking up membranes, exosomes, and/or ribonucleoprotein complexes inthe biological sample (e.g., a cell-free biological sample). Abiological sample that may be used to analyze and sequence RNAs usingmethods described herein may be, or may be derived from, a whole bloodsample, a plasma sample, a serum sample, a saliva sample, a cell culturemedia sample, a urine sample, an amniotic fluid sample, a mucus sample,a semen sample, a vaginal fluid sample, a sputum sample, a cerebrospinalfluid sample, a lymphatic fluid sample, an ocular fluid sample, a sweatsample, or a stool sample. In some embodiments, the above-mentionedsamples may be used directly as the biological sample in the methods. Inother embodiments, the above-mentioned samples may undergo a process toremove cells present in the samples in order to acquire cell-freebiological samples to be used in the methods. For example, a whole bloodsample may be processed to remove cells, e.g., white blood cells and redblood cells, to obtain a cell-free biological sample. Methods anddevices used for separating or removing cells from a biological sampleare known in the art and include, but are not limited to,plateletpheresis, sedimentation, and centrifugation.

A biological sample (e.g., a cell-free biological sample) may be lysedto break up membranes, exosomes, and/or ribonucleoprotein complexes. Insome embodiments, RNAs often exist in complex with proteins or inexosomes to be protected from RNase degradation. Exosomes arecell-derived vesicles that are present in biological samples. Exosomesmay be released from the cell when multivesicular bodies fuse with theplasma membrane or released directly from the plasma membrane. Themolecular components in exosomes include, e.g., RNAs, DNAs, andproteins. Ribonucleoprotein complexes are protein complexes that containRNAs, e.g., protein-RNA complexes. For example, RNAs that may becomplexed with proteins include, but are not limited to, lincRNAs (longintergenic noncoding RNAs), miRNAs (microRNAs), and snRNAs (smallnuclear RNAs). A biological sample (e.g., a cell-free biological sample)may be lysed using a lysis buffer to break up membranes and exosomes anddissociate ribonucleoprotein complexes. A lysis buffer may be anyaqueous solution that is capable of breaking open or lysing themembranes, exosomes, and/or ribonucleoprotein complexes in thebiological sample (e.g., a cell-free biological sample) to release theRNAs into the solution without degrading, fragmenting, or modifying theRNAs. In some embodiments, a lysis buffer used in the methods of theinvention may include, e.g., one or more detergents (e.g., NP-40), oneor more proteases, one or more RNase inhibitors (e.g., a solutioncontaining between about 5% w/v and about 15% w/v SDS (e.g., about 6%w/v, about 8% w/v, about 10% w/v, about 12% w/v, or about 14% w/v SDS;e.g., about 10% w/v SDS)), one or more redox agents (e.g., a DTT(dithiothreitol) solution), one or more salts, one or more bufferingagents, and/or one or more chelating agents. A lysis buffer may also beadjusted to or kept at a desired pH range (e.g., a pH of between 6 and 8(e.g., a pH of 6, 6.2, 6.4, 6.6, 6.8, 7, 7.2, 7.4, 7.6, 7.8, or 8, suchas a pH of 7.4) for the most efficient lysis and to provide a stableenvironment for the RNAs. Furthermore, depending on the type ofbiological sample, the detergents, proteases, RNase inhibitors, salts,buffering agents, chelating agents, and their respective amounts andconcentrations in the lysis buffer may be tailored to the specificbiological sample. The components of the lysis buffer described hereinmay be provided in admixture or separately added to the biologicalsample to form the lysis solution.

The biological sample (e.g., a cell-free biological sample) may becontacted with a lysis buffer to form a lysis solution. In someembodiments, the lysis solution may be mixed and incubated at roomtemperature for about 1 minute to about 10 minutes (e.g., about 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 minutes; e.g., about 5 minutes) for efficientmembrane and exosome lysis and ribonucleoprotein complex dissociation.In other embodiments, the lysis solution may be mixed and incubated in aheated environment at, e.g., between about 50° C. and about 60° C.(e.g., about 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58°C., or 59° C.; e.g., about 55° C.) for about 1 minute to about 10minutes (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes; e.g.,about 5 minutes) for efficient membrane and exosome lysis andribonucleoprotein complex dissociation, provided that the heatedenvironment does not degrade the RNAs.

Detergents in the lysis buffer enable the disruption and solublizationof membranes, exosomes, and proteins. Detergents are amphipathicmolecules containing both a nonpolar tail group having aliphatic oraromatic character and a polar head group. Detergents in the lysisbuffer may be nonionic, anionic, cationic, zwitterionic, or a mixturethereof. Examples of detergents that may be included in a lysis bufferused in methods of the invention include, but are not limited to, NP-40,Triton X 100, Triton X-114, Tween 20 (polysorbate 20), Tween 40, Tween60, Tween 80, 3 [(3 cholamidopropyl)dimethylammonio]-1-propanesulfonate(CHAPS), 3 [(3cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO),octyl glucoside, octyl thioglucoside, bile salts (e.g., cholate), andquaternary ammonium surfactants (e.g., cetyl trimethyl ammonium bromide(CTAB), tetradecyl trimethyl ammonium bromide (TTAB), ethyl trimethylammonium bromide (ETAB)). In some embodiments, the detergent in thelysis buffer is NP-40. The amount of detergent in a lysis buffer may beoptimized by one of skill in the art. In some embodiments, the lysisbuffer contains between 0.01% and 10% (e.g., about 0.025%, about 0.05%,about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%,about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%;e.g., about 4%) of a detergent (e.g., NP-40).

In some embodiments, the lysis buffer used in the methods of theinvention may include one or more proteases. A protease is an enzymethat breaks down proteins into smaller peptides and amino acids byproteolysis. Proteases may be classified according to the catalyticgroup involved in its active site. Examples of classes of proteasesinclude, but are not limited to, serine proteases (e.g., proteinase K,chymotrypsin, trypsin, elastase, plasmin, thrombin, acrosomal protease,complement C1, keratinase, collagenase, fibrinolysin, and cocoonase),cysteine proteases (e.g., papain, bromelain, cathepsin, calpain,caspase-1, sortase, TEV protease, and hepatitis C virus peptidase 2),threonine proteases, aspartic proteases, glutamic proteases,metalloproteases, and asparagine peptide lyases. In some embodiments ofthe methods described herein, the lysis buffer includes a serineprotease (e.g., proteinase K).

The lysis solution may further contain a buffering agent to prevent arapid change in pH of the solution. Examples of a buffering agentinclude, but are not limited to, tris(hydroxymethyl)aminomethane (Tris),citric acid, acetic acid, potassium phosphate, borate,N-Cyclohexyl-2-aminoethanesulfonic acid (CHES),tris(hydroxymethyl)methylamino-propanesulfonic acid (TAPS), bicine,tricine, 3-(N-Tris-(hydroxymethyl)methylamino)-2-hydroxypropanesulfonicacid (TAPSO), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES),2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid(TES), 3-(N-morpholino)propanesulfonic acid (MOPS),piperazine-N,N′-bis(2-ethanesulfonic acid (PIPES), cacodylate, and2-(N-morpholino)ethanesulfonic acid (MES). In some embodiments, thebuffering agent in the lysis buffer is Tris. A buffering agent maymaintain the pH of the lysis buffer at a pH of between 6 and 8 (e.g., apH of 6, 6.2, 6.4, 6.6, 6.8, 7, 7.2, 7.4, 7.6, 7.8, or 8, such as a pHof 7.4) for the most efficient membrane, exosome, and ribonucleoproteincomplex lysis and dissociation and to provide a stable environment forthe isolated RNAs. The lysis buffer used in methods of the invention mayalso contain additional reagents, such as chelating agents, reducingagents, stabilizers, organic or inorganic salts, metal ions, and/or pHindicators. A chelating agent, such as ethylenediaminetetraacetic acid(EDTA), may be included in the lysis buffer (e.g., as Tris-EDTA, knownalso as TE buffer).

Following membrane and exosome lysis and ribonucleoprotein complexdissociation, the sample may be processed to capture the RNAs by primerannealing. A primer mixture containing, e.g., poly(T) primers (e.g.,TTTTTT) that can hybridize to the poly(A) tail of the RNAs and aplurality of random primers (e.g., primers having six nucleotides; e.g.,hexamers), may be added to the sample to anneal to the RNAs. Theconditions for primer annealing may be tailored based on the length ofthe primers and the biological sample by one of skill in the art. Forexample, the sample may be incubated with the primer mix at about 70° C.for about 2 minutes for the primers to anneal to the RNAs.

Further, the sample may be processed to remove DNAs present in thesample. A DNase (e.g., DNase I or DNase II) may be added to the sampleto degrade DNAs by catalyzing the hydrolytic cleavage of phosphodiesterlinkages in the DNA backbone, thus degrading the DNAs. In someembodiments, the DNase used in the methods described herein is DNase I.In particular embodiments, an RNase inhibitor may be pre-mixed with aDNase-containing reagent and mixture may be added to the sample. AnRNase inhibitor may be a protein or small molecule compound used toinhibit RNA degradation by RNase. In particular embodiments, the RNaseinhibitor is a protein that binds to certain classes of ribonucleases(e.g., an RNase inhibitor that inhibits RNases A, B, and C). In someembodiments, the RNase inhibitor is a small molecule compound.

For example, a mixture containing a DNase-containing reagent and anRNase inhibitor may be added to the sample after membrane and exosomelysis and ribonucleoprotein complex dissociation. The resulting mixturemay be mixed and incubated at, e.g., 37° C. and/or 65° C., for a fewminutes, e.g., 5-10 minutes, for the enzymatic DNA degradation, e.g., byDNase I, to take place (e.g., incubation at 37° C. for 10 minutesfollowed by 65° C. for 5 minutes). In some embodiments, a higherincubation temperature (e.g., 65° C.) may be used to deactivate theDNase and/or the RNase inhibitor.

IV. Reverse-Transcription of RNAs to cDNAs

First Strand cDNA Synthesis

A complementary DNA (cDNA) is a DNA oligonucleotide synthesized from anRNA template in a reaction catalyzed by the enzyme reversetranscriptase. In some embodiments, a cDNA oligonucleotide also refersto a second DNA oligonucleotide generated based on a first cDNAoligonucleotide as the template. A reverse transcriptase operates on asingle-stranded RNA and generates its cDNA based on the pairing of RNAbase pairs (e.g., adenine, guanine, cytosine, and uracil) to their DNAcomplements (e.g., thymine, cytosine, guanine, and adenine,respectively). The sample containing the RNAs annealed with the primersas described above is used to synthesize the first strand cDNA. Forexample, a mixture containing a DNA polymerase I (e.g., an E. coli DNApolymerase I) and in some embodiments, also an RNase inhibitor (e.g., aprotein that inhibits RNases A, B, and C) may be added to the sample, sothe DNA polymerase I can use the RNA as a template and synthesize thecDNA by adding nucleotides to the 3′ end of the primers that areannealed to the RNA. The DNA polymerase I eventually generates aRNA-cDNA hybrid. The reaction conditions used to generate the RNA-cDNAhybrid may be designed and adjusted by one of skill in the art based on,e.g., the length of the RNA template, the type of biological sample, andthe concentration of the RNAs. For example, a thermocycler programmedto, e.g., 25° C. for 10 minutes, 40° C. for 5 minutes, and 70° C. for 10minutes, may be used to generate the RNA-cDNA hybrid.

First Strand cDNA Processing and RNA Removal

Once the RNA-cDNA hybrids are generated in the sample, the hybrids maybe fragmented to shorter strands and the RNA portion may be removed.Enzymes such as fragmentases may be used to generate shorter RNA-cDNAstrands. For example, a two-enzyme system including two fragmentases maybe used, in which one fragmentase generates single-strand nicks on onestrand of the RNA-cDNA hybrid and the other fragmentase cuts the otherstrand at locations corresponding to the nicks. The fragmentationreaction may be performed by adding the two fragmentases to the sampleand incubating the sample at, e.g., 37° C. for about 30 minutes. Theresulting products are short RNA-cDNA hybrids. Following thefragmentation reaction, the RNA portion of the hybrids may be degradedby adding a buffer rich in high metal ion (e.g., a buffer containing ahigh concentration of Zn²⁺) in combination with heat shock (e.g.,heating at 90° C. for about 2 minutes). In other embodiments, the RNAportion of the hybrids may be degraded by adding an RNase to the sample.Subsequent to RNA degradation, normal ionic strength of the sample maybe restored by adding a chelating agent, such asethylenediaminetetraacetic acid (EDTA), which can effectively bind tothe metal ions (e.g., Zn²⁺) used during RNA degradation.

After the RNAs are degraded, the sample now contains first strand cDNAfragments. To ensure that each cDNA fragment contains a 5′ phosphate anda 3′ hydroxyl, a T4 polynucleotide kinase may be added to the sample torepair the cDNA fragments. The T4 polynucleotide kinase catalyzes thetransfer of a γ-phosphate from ATP to the 5′ hydroxyl end of the cDNAfragments, thus, adding a 5′ phosphate to the cDNA fragment. Further, a3′ primer (e.g., a poly(G) (e.g., GGG) portion) may be added to the 3′end of the cDNA fragments using a terminal transferase, which aspecialized DNA polymerase that catalyzes the addition of nucleotides tothe 3′ end of a DNA molecule. A terminal transferase does not require atemplate to perform the reaction. At this stage, a plurality of shortcDNA oligonucleotides each containing a 3′ primer (e.g., a 3′ poly(G)portion (e.g., GGG)) is present in the sample.

Second Strand cDNA Synthesis

Each cDNA fragment containing a 3′ primer (e.g., a 3′ poly(G) portion(e.g., GGG)) may be used as a template to generate a second strand cDNAoligonucleotide. Note that the sequences of the second cDNAoligonucleotides are the same as the sequences of the RNAs, except thatthe uracils are replaced with thymidines. Also note that the sequencesof the first cDNA oligonucleotides are the same as the sequences of thegenomic DNAs, from which the RNAs are transcribed. In some embodiments,to generate the second strand cDNA oligonucleotide, the sample may beincubated with a primer that contains two parts: (1) a degradablesequence (e.g., a uracil DNA glycosylase recognition sequence) and (2) asequence complementary to the sequence of the 3′ primer (e.g., a poly(C)sequence (e.g., CCC)), as well as a reverse transcriptase (e.g., a MMLV(moloney murine leukemia virus) reverse transcriptase). The part (2) ofthe primer (e.g., the poly(C) sequence (e.g., CCC)) may hybridize withthe 3′ primer (e.g., the 3′ poly(G) portion (e.g., GGG)) on each of thefirst cDNA oligonucleotides. In some embodiments, the synthesis of thesecond strand cDNAs may proceed in two directions. In one instance, theMMLV reverse transcriptase may use the first cDNA oligonucleotide as atemplate to generate the second cDNA oligonucleotide. In anotherinstance, the MMLV reverse transcriptase may use the primer (e.g., theprimer containing the degradable sequence (e.g., the uracil DNAglycosylase recognition sequence) and the sequence complementary to thesequence of the 3′ primer (e.g., the poly(C) sequence (e.g., CCC))) asthe template to further elongate the first cDNA oligonucleotide. Asshown in FIG. 1, when the MMLV uses the primer as the template, thefirst cDNA oligonucleotide gets elongated at the 3′ end by the additionof a sequence that is complementary to the degradable sequence (e.g.,the uracil DNA glycosylase recognition sequence) of the primer. At thisstage, the sample contains a plurality of short, double-stranded cDNAoligonucleotides. Moreover, a T4 DNA polymerase may be added to thesample to remove any 3′ end overhangs in the double-stranded cDNAoligonucleotides by elongating the shorter strand to create a blunt 3′end in each double-stranded cDNA oligonucleotide. Further, a Klenow-exofragment enzyme may be added to the sample to ligate an adenine to the3′ end of each strand of the double-stranded cDNA oligonucleotides. Asshown in FIG. 1, at this stage, the sample contains a plurality ofshort, double-stranded cDNA oligonucleotides, in which eachdouble-stranded cDNA oligonucleotides contains an adenine at the 3′ endof each strand.

V. Adaptor Ligation, PCR Amplification, and Strand Selection

Each short, double-stranded cDNA oligonucleotide is additionally ligatedwith at least one adaptor. In some embodiments, each short,double-stranded cDNA oligonucleotide is additionally ligated with twoadaptors, one adaptor at each end of the oligonucleotide. In someembodiments, the two adaptors may be the same. In other embodiments, thetwo adaptors may be different. As shown in FIG. 1, each short,double-stranded cDNA oligonucleotide is additionally ligated with aforward adaptor and a reverse adaptor. As shown in FIG. 1, the forwardadaptor may include a degradable sequence (e.g., a uracil DNAglycosylase recognition sequence) that can be recognized by a uracil DNAglycosylase and a 3′ uracil overhang that specifically anneals to the 3′adenine on the first strand cDNA oligonucleotide. The reverse adaptormay include a 3′ thymine overhang that specifically anneals to the 3′adenine on the second strand cDNA oligonucleotide. The 3′ uraciloverhang of the forward adaptor and the 3′ thymine overhang of thereverse adaptor may allow specific strand selection downstream. As shownin FIG. 1, the desired ligation includes the forward adaptor beingligated to the 5′ end of the second strand cDNA oligonucleotide and thereverse adaptor being ligated to the 5′ end of the first strand cDNAoligonucleotide. One undesired ligation includes the forward adaptorbeing ligated to the 5′ of the first strand cDNA oligonucleotide and thereverse adaptor being ligated to the 5′ end of the second strand cDNAoligonucleotide. In some embodiments, the undesired ligation product,which may be a double-stranded cDNA oligonucleotide having thedegradable sequence (e.g., the uracil DNA glycosylase recognitionsequence) on both ends, may not be recognized during the downstream PCRamplification reaction. Thus, only the short, double-stranded cDNAoligonucleotides having different 5′ and 3′ ends may be amplified.

After adaptor ligation, the sample containing the adaptor-ligated,double-stranded cDNA oligonucleotides may be purified using variouscharge based nucleotide purification methods, such as solid-phaseseparation methods, e.g., chromatography. Purification of the sampleremoves undesired materials from the previous steps, e.g., any proteinseither from the original biological sample or added during sampleprocessing and nucleic acid debris (e.g., degraded RNAs and DNAs),before the adaptor-ligated, double-stranded cDNA oligonucleotidesundergo PCR amplification and subsequent sequencing. Many availablecharge based nucleotide purification methods rely on the interactionsbetween the oligonucleotide and the solid phase based on their charges.In some embodiments, a charge based nucleotide purification method mayuse silica based columns or silica coated beads (e.g., silica coatedmagnetic beads). For example, solid-phase separation methods utilize theattractive interactions between nucleic acid molecules and silicasurfaces under optional salt concentrations and pH. In some embodiments,the sample may be mixed with beads, such as magnetic beads coated withsilica on their surfaces. Once the adaptor-ligated, double-stranded cDNAoligonucleotides bind to the magnetic beads, the beads can be separatedfrom the aqueous solution using a magnetic separator. The isolatedadaptor-ligated, double-stranded cDNA oligonucleotides can subsequentlybe eluted from the magnetic beads using water or buffered water (e.g.,tris-acetate buffered water). The ratio of nucleic acids to magneticbeads may be optimized to improve nucleic acid yield. Magnetic beadsdesigned to isolate nucleic acids are described in, e.g., Smith et al.,J. Clin. Microbiol., 41:2440-2443, 2003, Miszczak et al., J. Clin.Microbiol., 49:3694-3696, 2011, and Yang et al., J. Virol. Methods,171:195-199, 2011. Various magnetic beads are also commerciallyavailable, e.g., AMPure® beads, MagJET® beads, and Magesil Blue® beads.In other embodiments, the sample may be loaded onto a column containingsilica as the solid phase. The supernatant can be removed either bygravity or centrifugation (in the case of spin-columns). The isolatedadaptor-ligated, double-stranded cDNA oligonucleotides can then beeluted from the column using water or buffered water (e.g., tris-acetatebuffered water).

The purified sample may undergo a first stage of PCR amplification. Asshown in FIG. 1, during the first stage of PCR amplification, theforward and reverse primers are designed such that only theadaptor-ligated, double-stranded cDNA oligonucleotides from the desiredadaptor ligation reaction may be amplified. Further to the first stageof PCR amplification, the PCR product may be purified using magneticbeads (e.g., AMPure® beads). The PCR product contains double-strandedcDNA oligonucleotides in which the strand having the degradable sequence(e.g., the uracil DNA glycosylase recognition sequence) at the 5′ end(e.g., the second strand cDNA oligonucleotide) may be degraded by theenzyme uracil DNA glycosylase. The remaining strand has the sequence ofthe first strand cDNA oligonucleotide, which is also the sequence of thegenomic DNA used to transcribe the RNA, and is thus selected to undergoa second stage of PCR amplification.

Prior the second stage of PCR amplification, cDNA oligonucleotideshaving the sequences of ribosomal RNAs (rRNAs) may be removed using anrRNA primer that anneals specifically to cDNA oligonucleotides that arederived from rRNAs. Once these cDNA oligonucleotides are amplified usinga Taq DNA polymerase, an endonuclease cleavage site integrated in thereverse adaptor is exposed. Using specific endonucleases, the reverseadaptor may be cleaved to render the resulting double-stranded cDNAunable to undergo further rounds of PCR amplification. Examples ofendonucleases include, but are not limited to, EcoRI, EcoRII, BamHI,HindIII, TaqI, NotI, HinFI, Sau3AI, PvuII, SmaI, HaeIII, HgaI, AluI,EcoRV, EcoP15I, KpnI, PstI, SacI, SalI, ScaI, SpeI, SphI, StuI, andXbaI. Thus, at this stage, the sample contains cDNA oligonucleotideshaving the same sequences as those of the genomic DNAs and is free ofany cDNA oligonucleotides corresponding to rRNAs. Another round of PCRamplification followed by purification on magnetic beads generate a cDNAlibrary that is ready for sequencing.

VI. Sequencing and Genome Mapping

The cDNA library generated from the RNAs may be sequenced to detectvarious RNA genome equivalents that were present in the originalbiological sample. The cDNA library may be sequenced using any availablesequencing techniques. For example, sequencing methods include classicalpolymerase-mediated enzymatic methods such as Sanger dideoxy sequencing,as well as capillary based implementations of Sanger sequencing andautomated implementations of Sanger sequencing. These commerciallyavailable systems for Sanger sequencing include, e.g., 1-CapillarySequencers, 4-Capillary Sequencers, 16-Capillary Sequencers,48-Capillary Sequencers, 96-Capillary Sequencers, and the ABI Prism®3700 series DNA analyzers. Many sequencing approaches include an invitro cloning step to generate many copies of each individual molecule.For example, in emulsion PCR individual nucleic acid molecules areisolated along with primer-coated beads in aqueous bubbles within an oilphase. A polymerase chain reaction (PCR) then coats each bead withclonal copies of the isolated library molecule and these beads aresubsequently immobilized for later sequencing. In other cases, surfacemethods of clonal amplification have been developed, for example, by theuse of bridge PCR where fragments are amplified upon primers attached toa solid surface. These methods produce many physically isolatedlocations which each contain many copies of a single fragment.Next-generation sequencing methods may also be used to sequence the cDNAlibrary. Examples of next-generation sequencing methods include, but arenot limited to, single-molecule real-time sequencing, ion semiconductorsequencing, pyrosequencing, sequencing by synthesis, sequencing bybridge amplification, sequencing by ligation, nanopore sequencing, chaintermination sequencing, massively parallel signature sequencing, polonysequencing, heliscope single molecule sequencing, and oligonucleotideextension sequencing. Various sequencing technologies are described in,e.g., Goodwin et al., Nature Review Genetics 17:333, 2016; Buermans andDunnen, Biochim. Biophys. Acta 1842: 1932, 2014; Heather and Chain,Genomics 107:1, 2016; and Levy and Myers, Annu. Rev. Genom. Hum. Genet.17:95, 2016, which are all incorporated by reference herein.

The methods described herein may also include subjecting the cDNAlibrary to digital counting and analysis. The number of amplifiedsequences for each transcript in the amplified sample can be quantitatedthrough sequence reads (e.g., one read per amplified strand).Quantitation during sequencing may allow for the detection andquantitation for each transcript present in the biological sample (e.g.,a cell-free biological sample) containing RNAs. The methods describedherein use only a small amount of biological sample (e.g., less than orequal to about 1 mL) to obtain sequencing reads that can be used todetect the RNA genome equivalents present in the biological sample, aswell as mapping the RNA genome equivalents to different categories ofRNAs.

Once the cDNA library is sequenced, the sequences may be mapped to theirRNA genome equivalents. Various gene mapping and sequence alignmenttools and databases are available, e.g., BLAST, FASTA, Genoogle, HMMER,USEARCH, ScalaBlast, and Genome Compiler. Each tool or database may betailored to the specific goals of the gene mapping, e.g., filters may beset to search within the genes belonging to a particular tissue ororgan, as well as the length of the target gene pool. Gene annotationsmay also be set to search within the genome of a particular organism orspecies (e.g., ENSEMBL gene annotation HG38). As demonstrated inExamples 1-3, using only 45 μL of the cell-free biological sample asinput, the methods of the invention are able to detect 95% ofprotein-coding genes having ENSEMBL gene annotation HG38 (e.g., lincRNA,miRNA, and snRNA) and 176 tissue-specific genes for the brain, bonemarrow, and the peripheral nervous system (PNS). Depending on thebiological sample, in some embodiments, the methods described herein maydetect at least about 100 different genes while using less than or equalto about 1 mL (e.g., less than or equal to about 500 μL, less than orequal to about 250 μL, less than or equal to about 100 μL, less than orequal to about 75 μL, less than or equal to about 50 μL, less than orequal to about 25 μL, less than or equal to about 20 μL, less than orequal to about 15 μL, or less than or equal to about 10 μL) of thebiological sample. Categories of RNAs that may be detected using methodsof the invention include, but are not limited to, lincRNAs, miRNAs,snRNAs, ncRNAs, nmRNAs, sRNAs, smnRNAs, tRNAs, mRNAs, pcRNAs, rRNAs, 5SrRNAs, 5.8S rRNAs, SSU rRNAs, LSU rRNAs, NoRC RNAs, pRNAs, 6S RNAs, SsrSRNAs, aRNAs, asRNAs, asmiRNAs, crRNAs, tracrRNAs, DD RNAs, diRNAs,dsRNAs, endo-siRNAs, exRNAs, gRNAs, hc-siRNAs, hcsiRNAs, hnRNAs, RNAi,lncRNAs, mrpRNAs, nat-siRNAs, natsiRNAs, OxyS RNAs, piRNAs, qiRNAs,rasiRNAs, scaRNAs, scnRNAs, scRNAs, scRNAs, SgrS RNAs, shRNAs, siRNAs,SL RNAs, SmY RNAs, snoRNAs, snRNP, RP RNAs, ssRNAs, stRNAs, tasiRNAs,tmRNAs, uRNAs, vRNAs, vtRNAs, Xist RNAs, Y RNAs, pre-mRNAs, and circRNAs(e.g., lincRNAs, miRNAs, and snRNAs).

VII. Devices

In some embodiments, one or more steps of constructing a cDNA libraryand detecting the RNA genome equivalents in the cDNA library may beautomated using one or more automated sample handling devices (e.g., oneor more automated liquid or fluid handling devices). Automated devicesand procedures may be used to deliver reaction reagents, including oneor more of the following: biological samples, buffers, enzymes, primers,salts, and any other suitable agents. Automated devices and proceduresalso may be used to control the reaction conditions. For example, anautomated thermal cycler may be used to control reaction temperaturesand any temperature cycles that may be used. In some embodiments, ascanning laser may be automated to provide one or more reactiontemperatures or temperature cycles suitable for incubatingpolynucleotides and/or various enzymes (e.g., polymerases, ligases, andproteases). Similarly, subsequent analysis of the cDNA libraryconstructed from the RNAs present in the sample may be automated. Forexample, sequencing may be automated using a sequencing device andautomated sequencing protocols as described above. In some embodiments,one or more of the devices or device components described herein may becombined in a system (e.g., a robotic system) or in a micro-environment(e.g., a micro-fluidic reaction chamber). Assembly reaction mixtures(e.g., liquid reaction samples) may be transferred from one component ofthe system to another using automated devices and procedures (e.g.,robotic manipulation and/or transfer of samples and/or samplecontainers, including automated pipetting devices, micro-systems, etc.).The system and any components thereof may be controlled by a controlsystem.

Accordingly, the steps of the methods described herein (e.g., those inExample 2) and/or aspects of the devices described herein may beautomated using, for example, a computer system (e.g., a computercontrolled system). A computer system on which aspects of the technologyprovided herein can be implemented may include a computer for any typeof processing (e.g., sequence analysis and/or automated device controlas described herein). In some embodiments, a computer system may includetwo or more computers. For example, one computer may be coupled, via anetwork, to a second computer. One computer may perform sequenceanalysis. The second computer may control one or more of the automatedsynthesis and assembly devices in the system. In other embodiments,additional computers may be included in the network to control one ormore of the analysis or processing acts. Each computer may include amemory and processor. The computers can take any form, as the aspects ofthe methods provided herein are not limited to being implemented on anyparticular computer platform. Similarly, the network can take any form,including a private network or a public network. Display devices can beassociated with one or more of the devices and computers. Alternatively,or in addition, a display device may be located at a remote site andconnected for displaying the output of an analysis in accordance withthe technology provided herein. Connections between the differentcomponents of the system may be via wire, optical fiber, wirelesstransmission, satellite transmission, any other suitable transmission,or any combination of two or more of the above.

EXAMPLES

The present invention will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes, and are not intended to limit the invention in any manner.Those of skill in the art will readily recognize a variety ofnoncritical parameters which can be changed or modified to yieldessentially the same results.

Example 1 Sample Collection

Ordinary serum collection: A 10 mL whole blood sample was drawn intoserum separator tubes. No anticoagulants were added. The whole bloodsample was given 15-30 minutes to clot at room temperature with nodisruptions. The sample was then centrifuged in refrigerated conditionsfor 10 minutes at 1000-2000 g. After centrifugation, the liquidsupernatant, which contained serum, was transferred to cleanpolypropylene tubes using a Pasteur pipette. Multiple 1 mL aliquots weremade and were stored at −80° C. The process was finished within the sameday as the whole blood sample was collected to avoid potentialdegradation of proteins or nucleic acids in serum.

Fingertip serum collection: The collection site, usually on the tip ofthe left ring finger, was first cleaned with an alcohol wipe and thenpunctured with a lancet. At least 40 μL of blood was collected by gentlytouching the blood droplet formed at the puncture site with a plaincapillary tube. Blood flew into the capillary tube due to capillaryeffect. The capillary tube was then sealed and left undisturbed for 30minutes under room temperature to allow blood coagulation. The capillarytube with sample was then centrifuged under refrigerated conditions for10 minutes at 1000 g and serum was harvested as supernatant aftercentrifugation. The harvested serum was made into aliquots and stored at−80° C.

Cell-free saliva collection: Firstly, a saliva collection swab was usedto collect whole saliva from an individual. A swab was placed in themouth under the tongue for at least 5 minutes, and then inserted into asyringe barrel for downstream processing. Secondly, cells were removedby either squeezing whole saliva out of the swab and pressing it throughan attached 0.2 μm PES syringe filter or squeezing whole saliva out ofthe swab into a centrifuge tube and precipitating cells withrefrigerated centrifugation at 1500 g for 15 minutes. The filtrate outof the syringe filter and supernatant after centrifugation wereconsidered as cell-free saliva. The harvested cell-free saliva was madeinto aliquots and stored at −80° C.

IVF culture media sample collection: IVF cell culture media was usuallyprovided cell-free and as aqueous droplets immersed in culture oil(e.g., OVOIL) in a petri dish. Firstly, the culture media droplets,having a typical volume of 30-50 μL, were transferred to centrifugetubes with pipettes. Brief spin down was then applied to separate anyoil phase carry over to the top and the aqueous phase at the bottom wasaspirated out as IVF culture media into a new centrifuge tube. Theharvested sample was made into aliquots and stored at −80° C.

Ordinary plasma collection: A 10 mL whole blood sample was drawn intoanticoagulant-treated blood collection tubes using anticoagulants suchas heparin, EDTA or citrate-acid. The sample was then centrifuged inrefrigerated conditions for 10 minutes at 1000-2000 g. Aftercentrifugation, the liquid supernatant, which was plasma, wastransferred to clean polypropylene tubes using a Pasteur pipette.Multiple 1 mL aliquots were made and stored at −80° C.

Fingertip plasma collection: The collection site, usually on the tip ofthe left ring finger, was first cleaned with an alcohol wipe and thenpunctured with a lancet. At least 40 μL of blood was collected by gentlytouching the blood droplet formed at puncture site using a capillarytube coated with anticoagulant (e.g., heparin, EDTA, or citrate acid).Blood flew into the capillary tube due to capillary effect. Thecapillary tube was then sealed and centrifuged under refrigeratedconditions for 10 minutes at 1000 g and plasma was harvested assupernatant after centrifugation. The harvested plasma was made intoaliquots and stored at −80° C.

Example 2 Library Construction Procedures

Steps (a) to (o) below describe the construction of a cDNA library fromthe RNAs in the cell-free biological sample. The compositions andfunctions of the reagents and kits used in these steps are listed inTable 1.

TABLE 1 Composition or Commercial Reagent or Kit Catalog No. FunctionLysis Buffer Nuclease-free water, 10 nM Lyses, degrades, and Tris HCl,10% w/v SDS, 4% dissociates membranes, w/v NP-40 exosomes, andribonucleoproteins bound to the RNAs Dithiothreitol (DTT) Solution 11.5mM DTT Prevents oxidation RNase Inhibitor NEB #M0314 Prevents RNAdegradation by RNase First Strand Primer Mix A mixture of random primersServes as primers for cDNA (e.g., hexamers) and oligo-dT reversetranscription from primers RNAs DNase Buffer 3 mM MgCl₂ (reaction conc)Degrades any DNA present in 1 mM DTT (reaction conc) the biologicalsample HL-dsDNase ArcticZymes #70800-201 Degrades any DNA present in thebiological sample First Strand Buffer Mix NEB #M0209 Buffer for E. coliDNA polymerase I First Strand Enzyme Mix NEB #M0209 (E. coli DNA E. coliDNA Polymerase I used Polymerase I) for first strand cDNAoligonucleotide synthesis with first strand primer mix cDNA ProcessingEnzyme I NEB #M0348 Enzyme to fragment DNA- (Fragmentase I) RNA hybridgenerated after first strand cDNA synthesis cDNA Processing Enzyme IINEB #M0348 Enzyme to fragment DNA- (Fragmentase II) RNA hybrid generatedafter first strand cDNA synthesis cDNA Processing Reagent I Zinc ion(Zn²⁺) Buffer rich in heavy metal ion to facilitate RNA degradation cDNAProcessing Reagent II EDTA Buffer rich in EDTA to restore normal ionicstrength cDNA Processing Reagent III NEB #M0201 Buffer for T4Polynucleotide Kinase cDNA Processing Reagent IV NEB #M0315 Buffer forTerminal Transferase cDNA Processing Enzyme III NEB #M0201 (T4 T4Polynucleotide Kinase, Polynucleotide Kinase) repairs damaged 3′ and 5′ends and ensures that there are phosphate groups on 5′ ends and hydroxylgroups on 3′ ends cDNA Processing Enzyme IV NEB #M0315 (TerminalTerminal Transferase, adds 3 Transferase) guanines to 3′ end of thefirst strand cDNA Second Strand Buffer Mix 50 mM Tris-HCl (pH 8.3), 75Buffer for MMLV Reverse mM KCl, 3 mM MgCl₂, 10 Transcriptase mM DTTSecond Primer Mix Second strand primer base pairing with the 3 guanineson first strand cDNA 3′ end Second Strand Enzyme Mix Propspec #ENZ-310(MMLV Reverse transcriptase used here Reverse Transcriptase) for secondstrand cDNA synthesis, using first strand cDNA as template. End RepairEnzyme Mix I NEB #M0203 (T4 DNA T4 DNA Polymerase, trims Polymerase)cDNA to blunt end End Repair Enzyme Mix II NEB #M0212 (Klenow exo-Klenow exo- Fragment to trim Fragment) cDNA to blunt end and add adenine(A) to 3′ end of the two strands of cDNA Ligation Enzyme Mix NEB #M0202(T4 DNA T4 DNA Ligase, ligate adaptor Ligase) to double-strand cDNAAdaptor Inventory Barcoded adaptors for PCR amplification and multiplexsequencing Ligation Buffer Mix NEB #M0202 Buffer for T4 DNA LigaseNuclease-free Water Ambion #AM9916 Nuclease-free water AMPure beadsBeckman Coulter #A63987 Nucleotide purification on beads DNAResuspension Buffer 10 mM TRIS-Acetate, pH 8.0, Buffer used for DNAstorage reagent grade water; Or, TE media or elution media (from Buffer(10 mM Tris-Acetate magnetic beads) pH 8.0, 1 mM EDTA) AmplificationBuffer Mix NEB #E5000 Buffer for Taq DNA Polymerase, also contains dNTPAmplification Primer Mix I Primers base-pairing with adaptor foramplification, with the “reverse primer” consists of dUTP instead ofthymine Amplification Enzyme Mix NEB #E5000 (Taq DNA Taq DNA Polymerase,used Polymerase) for PCR amplification rRNA Depletion Primer Mix Primerthat base-pairs with specific human rRNA region Strand Selection EnzymeMix NEB #M0280 (uracil DNA Uracil DNA glycosylase glycosylase) (UDG),degrades the specific cDNA strand containing dUTP or a uracil DNAglycosylase recognition sequence Adaptor Cleavage Enzyme RestrictionEndonuclease Restriction endonuclease to Mix cleave adaptor off duringrRNA depletion so that cDNA strands carrying rRNA information is notamplifiable Amplification Primer Mix II Primers base-pairing withadaptor for amplification Double-strand DNA Thermo Fisher #32851Quantification tool for cDNA quantification kit A concentration inconstructed library Double-strand DNA gel Aligent #5067-4626 ExaminecDNA size electrophoresis array kit B distribution in constructedlibrary

a. Exosome Lysis and Nucleoprotein Dissociation

-   1. Thaw unprocessed serum samples from Example 1 on ice. Load 7 μL    of the unprocessed serum sample to each well of a PCR strip or PCR    plate.-   2. Take “Lysis Buffer” out of stock, vortex to mix, spin down, and    keep at room temperature. Take “DTT Solution” out from −20° C.    storage, thaw at room temperature, vortex to mixt, spin down, and    keep at room temperature. Take “RNase Inhibitor” out from −20° C.    storage, flick gently to mix, spin down, and put on ice.-   3. Prepare the lysis cocktail by mixing the following components    (the volume for each component is for one lysis reaction): Lysis    Buffer: 2.8 μL, DTT Solution: 1.7 μL, and RNase Inhibitor: 0.5 μL.    Mix the components thoroughly by pipetting up and down at least 10    times with 70% of total volume. Then spin down and put the lysis    cocktail on ice. Use immediately.-   4. Add 5 μL of the lysis cocktail from step (a)(3) to the 7 μL    unprocessed serum sample from step (a)(1) for a final volume of 12    μL. Mix thoroughly by pipetting up and down for at least 10 times.    Incubate at room temperature for 5 minutes. Proceed immediately to    the next step of primer annealing and DNA digestion or store the    mixture at −80° C.

b. Primer Annealing and DNA Digestion

-   1. Pre-warm thermal cycler to run Program 1 (70° C. for 2 min, hold    at 4° C.). Take “DNase Buffer” and “First Strand Primer Mix (mixture    of random and oligo-dT primer)” out from −20° C. storage, thaw under    room temperature, vortex to mix, spin down, and keep on ice.-   2. Prepare the primer annealing cocktail by mixing the following    components (the volume for each component is for one annealing    reaction): DNase Buffer: 1 μL and First Strand Primer Mix: 2.6 μL.    Mix thoroughly by pipetting up and down at least 10 times with 70%    of total volume. Then spin down and put on ice. Use immediately.-   3. Add 3.6 μL of the primer annealing cocktail from step (b)(2) to    each sample tube in step (a)(4) for a total volume of 15.6 μL. Mix    thoroughly by pipetting up and down for at least 10 times. Spin down    and place on ice.-   4. Place the tube from step (b)(3) in the pre-warmed thermal cycler    and start Program 1 (70° C. for 2 min, hold at 4° C.). Remove the    tube from the thermal cycler, spin down the condensation, and place    the tube on ice. Proceed to the next step.-   5. Pre-warm thermal cycler to run Program 2 (37° C. for 10 min,    65° C. for 5 min, hold at 4° C.). Take “HL-dsDNase” and “RNase    Inhibitor” out from −20° C. storage, flick gently to mix, spin down,    and keep on ice.-   6. Prepare a DNA digestion cocktail by mixing the following    components (the volume for each component is for one digestion    reaction): HL-dsDNase: 2 μL and RNase Inhibitor: 0.2 μL. Mix    thoroughly by pipetting up and down at least 10 times with 70% of    total volume. Then spin down and put on ice. Use immediately.-   7. Add 2.2 μL of the DNA digestion cocktail from step (b)(6) to each    sample tube from step (b)(4) for a total volume of 17.8 μL. Mix    thoroughly by pipetting up and down for at least 10 times. Spin down    and place on ice.-   8. Place the tube in the pre-warmed thermal cycler and start Program    2 (37° C. for 10 min, 65° C. for 5 min, hold at 4° C.). Remove the    tube out of the thermal cycler, spin down the condensation, and    place on ice, proceed to the next step of first strand cDNA    synthesis.

c. First Strand cDNA Synthesis

-   1. Pre-warm thermal cycler to run Program 3 (25° C. for 5 min,    40° C. for 30 min, 70° C. for 10 min, hold at 4° C.). Take “First    Strand Buffer Mix (buffer for E. coli DNA polymerase I)” out from    −20° C. storage, thaw under room temperature, vortex to mix, spin    down, and keep on ice. Take “First Strand Enzyme Mix (E. coli DNA    Polymerase I)” and “RNase Inhibitor” out from −20° C. storage, flick    gently to mix, spin down, and keep on ice.-   2. Prepare a first strand synthesis cocktail by mixing the following    components (the volume for each component is for one synthesis    reaction): First Strand Buffer Mix: 1 μL, First Strand Enzyme Mix: 1    μL and RNase Inhibitor 0.2 μL. Mix thoroughly by pipetting up and    down at least 10 times with 70% of total volume. Then spin down and    put on ice. Use immediately.-   3. Add 2.2 μL of the first strand synthesis cocktail from step    (c)(2) to each sample tube from step (b)(8) for a total volume of 20    μL. Mix thoroughly by pipetting up and down for at least 10 times.    Spin down and place on ice.

d. cDNA Processing

-   1. Pre-warm thermal cycler to run Program 4 (37° C. for 30 min, hold    at 4° C.). Take “cDNA Processing Enzyme I (Fragmentase I)” and “cDNA    Processing Enzyme II (Fragmentase II)” out from −20° C. storage,    flick gently to mix, spin down, and keep on ice. Take “cDNA    Processing Reagent I (buffer rich in heavy metal ion to facilitate    RNA degradation)”, “cDNA Processing Reagent II (buffer rich in EDTA    to restore normal ionic strength)”, “cDNA Processing Reagent III    (buffer for T4 Polynucleotide Kinase)” and “cDNA Processing Reagent    IV (buffer for Terminal Transferase)” out from −20° C. storage, thaw    under room temperature, vortex to mix, spin down, and keep on ice.-   2. Prepare cDNA fragmenting cocktail by mixing the following    components (the volume for each component is for one fragmenting    reaction): cDNA Processing Enzyme I: 0.5 μL and cDNA Processing    Enzyme II: 0.5 μL. Mix thoroughly by pipetting up and down at least    10 times with 70% of total volume. Then spin down and put on ice.    Use immediately.-   3. Add 1 μL of the cDNA processing cocktail to each sample tube from    step (c)(3) for a total volume of 21 μL. Mix thoroughly by pipetting    up and down for at least 10 times. Spin down and place on ice.-   4. Place the tube in the pre-warmed thermal cycler and start Program    4 (37° C. for 30 min, hold at 4° C.). Remove the tube out of the    thermal cycler, spin down the condensation, and place on ice.    Proceed to the next step immediately.-   5. Pre-warm thermal cycler to run Program 5 (90° C. for 20 min, hold    at 4° C.).-   6. Add 2 μL of “cDNA Processing Reagent I (buffer rich in heavy    metal ion to facilitate RNA degradation)” to each sample tube from    step (d)(4) for a total volume of 23 μL. Mix thoroughly by pipetting    up and down for at least 10 times. Spin down and place on ice.-   7. Place the tube in the pre-warmed thermal cycler and start Program    5 (90° C. for 20 min, hold at 4° C.). Remove the tube out of the    thermal cycler, spin down the condensation, and place on ice.    Proceed to the next step immediately.-   8. Add 2 μL of “cDNA Processing Reagent II (buffer rich in EDTA to    restore normal ionic strength)” to each sample tube from step (d)(7)    for a total volume of 25 μL. Mix thoroughly by pipetting up and down    for at least 10 times. Spin down and place on ice.-   9. Pre-warm thermal cycler to run Program 6 (37° C. for 30 min,    70° C. for 10 min, hold at 4° C.). Take “cDNA Processing Enzyme III    (T4 Polynucleotide Kinase to repair damaged 3′ and 5′ ends)” out    from −20° C. storage, flick gently to mix, spin down, and keep on    ice.-   10. Prepare a cDNA repair cocktail by mixing the following    components (the volume for each component is for one repair    reaction): cDNA Processing Reagent III: 1 μL and cDNA Processing    Enzyme III: 1μL. Mix thoroughly by pipetting up and down at least 10    times with 70% of total volume. Then spin down and put on ice. Use    immediately.-   11. Add 2 μL of the cDNA repair cocktail to each sample tube from    step (d)(8) for a total volume of 27 μL. Mix thoroughly by pipetting    up and down for at least 10 times. Spin down and place on ice.-   12. Place the tube in the pre-warmed thermal cycler and start    Program 6 (37° C. for 30 min, 70° C. for 10 min, hold at 4° C.).    Remove the tube out of the thermal cycler, spin down the    condensation, and place on ice. Proceed to the next step    immediately.-   13. Pre-warm thermal cycler to run Program 7 (37° C. for 30 min,    75° C. for 20 min, hold at 4° C.). Take “cDNA Processing Enzyme IV    (Terminal Transferase to add 3 guanine to 3′ end of the first strand    cDNA)” out from −20° C. storage, flick gently to mix, spin down, and    keep on ice.-   14. Prepare a cDNA transferase cocktail by mixing the following    components (the volume for each component is for one transferase    reaction): cDNA Processing Reagent IV: 7 μL and cDNA Processing    Enzyme IV: 1 μL. Mix thoroughly by pipetting up and down at least 10    times with 70% of total volume. Then spin down and put on ice. Use    immediately.-   15. Add 8 μL of the cDNA transferase cocktail to each sample tube    from step (d)(12) for a total volume of 35 μL. Mix thoroughly by    pipetting up and down for at least 10 times. Spin down and place on    ice.-   16. Place the tube in the pre-warmed thermal cycler and start    Program 7 (37° C. for 30 min, 75° C. for 20 min, hold at 4° C.).    Remove the tube out of the thermal cycler, spin down the    condensation, and place on ice. Proceed to the next step of second    strand cDNA synthesis immediately.

e. Second Strand Synthesis

-   1. Pre-warm thermal cycler to run Program 8 (25° C. for 15 min,    37° C. for 15 min, 70° C. for 10 min, hold at 4° C.). Take “Second    Strand Buffer Mix (buffer for MMLV Reverse Transcriptase)” and    “Second Primer Mix (second strand primer base paring with the 3    guanines on first strand cDNA 3′ end)” out from −20° C. storage,    thaw at room temperature, vortex to mix, spin down, and keep on ice.    Take “Second Strand Enzyme Mix (MMLV Reverse Transcriptase)” out    from −20° C. storage, flick gently to mix, spin down, and keep on    ice.-   2. Prepare a second strand synthesis cocktail by mixing the    following components (the volume for each component is for one    synthesis reaction): Second Strand Buffer Mix: 3.5 μL, Second Strand    Primer Mix: 3.5 μL, and Second Strand Enzyme Mix: 1 μL. Mix    thoroughly by pipetting up and down at least 10 times with 70% of    total volume. Then spin down and put on ice. Use immediately.-   3. Add 8 μL of the second strand synthesis reaction to each sample    tube from step (d)(15) for a volume of 43 μL. Mix thoroughly by    pipetting up and down for at least 10 times. Spin down and place on    ice.-   4. Place the tube in the pre-warmed thermal cycler and start Program    8 (25° C. for 15 min, 37° C. for 15 min, 70° C. for 10 min, hold at    4° C.). Remove the tube out of the thermal cycler, spin down the    condensation, and place on ice. Proceed to the next step    immediately.

f. End Repair

-   1. Pre-warm thermal cycler to run Program 9 (25° C. for 30 min,    70° C. for 10 min, hold at 4° C.). Take “End Repair Enzyme Mix I (T4    DNA Polymerase to trim cDNA to blunt end)” and “End Repair Enzyme    Mix II (Klenow exo-Fragment to trim cDNA to blunt end and add    adenine to 3′ end of the two strands of cDNA)” out from −20° C.    storage, flick gently to mix, spin down, and keep on ice.-   2. Prepare an end repair cocktail by mixing the following components    (the volume for each component is for one repair reaction): End    Repair Enzyme Mix I: 1 μL and End Repair Enzyme Mix II: 1 μL. Mix    thoroughly by pipetting up and down at least 10 times with 70% of    total volume. Then spin down and put on ice. Use immediately.-   3. Add 2 μL of the end repair cocktail to each sample tube from step    (e)(4) for a total volume of 45 μL. Mix thoroughly by pipetting up    and down for at least 10 times. Spin down and place on ice.-   4. Place the tube in the pre-warmed thermal cycler and start Program    9 (25° C. for 30 min, 70° C. for 10 min, hold at 4° C.). Remove the    tube out of the thermal cycler, spin down the condensation, and    place on ice. Proceed to the next step of “Adaptor Ligation”    immediately or store the mixture at −20° C.

g. Adaptor Ligation

-   1. Pre-warm thermal cycler to run Program 10 (25° C. for 30 min,    70° C. for 10 min, hold at 4° C.). Take “Ligation Enzyme Mix (T4 DNA    Ligase)” out from −20° C. storage, flick gently to mix, spin down,    and keep on ice. Take “Adaptor Inventory (barcoded adaptors for PCR    amplification and multiplex sequencing)” out from −20° C. storage,    thaw under room temperature, vortex to mix, spin down, and keep on    ice. Take “Ligation Buffer Mix (buffer for T4 DNA Ligase)” and    “Nuclease-free Water” out from −20° C. storage, thaw under room    temperature, vortex to mix, spin down, and keep on ice. Take “AMPure    beads (Magnetic beads for nucleotide purification)” out from 4° C.    storage and “DNA Resuspension Buffer (10 mM TRIS-Acetate, pH 8.0,    reagent grade water or, TE Buffer [10 mM Tris-Acetate pH 8.0, 1 mM    EDTA])” out from −20° C. storage, equilibrate, and thaw at room    temperature for at least 30 minutes before the next step of “Adaptor    Ligation Purification.”-   2. Add 3.25 μL of different appropriate barcoded adaptors to each of    the tubes from step (0(4) for a total volume of 48.25 μL. Mix    thoroughly by pipetting. Then spin down and place on ice.-   3. Prepare a ligation cocktail by mixing the following components    (the volume for each component is for one ligation reaction):    Ligation Buffer Mix: 13 μL, Ligation Enzyme Mix: 2 μL, and    Nuclease-free Water: 1.75 μL. Mix thoroughly by pipetting up and    down at least 10 times with 70% of total volume. Then spin down and    put on ice. Use immediately.-   4. Add 16.75 μL of the ligation cocktail to each sample tube from    step (g)(2) for a total volume of 65 μL. Mix thoroughly by pipetting    up and down for at least 10 times. Spin down and place on ice.-   5. Place the tube in the pre-warmed thermal cycler and start Program    10 (25° C. for 30 min, 70° C. for 10 min, hold at 4° C.). Remove the    tube out of the thermal cycler, spin down the condensation, and    place on ice. Proceed to the next step of “Adaptor Ligation    Purification” immediately or store the mixture at −20° C.

h. Adaptor Ligation Purification (Conducted at Room Temperature)

-   1. Make sure that “AMPure beads” and “DNA Resuspension Buffer” are    thawed completely and are equilibrated to room temperature. This    usually takes at least 30 minutes. Vortex “AMPure beads” and “DNA    Resuspension Buffer” to resuspend and mix. Leave the reagents at    room temperature. Do not spin down beads. Take “Nuclease-free Water”    out from −20° C. storage, thaw under room temperature, vortex to    mix, spin down, and keep at room temperature. Make sure “AMPure    Beads” are fully suspended before use. Prepare 70% ethanol freshly.-   2. Add 35 μL “Nuclease-free Water” to each tube from step (g)(5) for    a volume of 100 μL. Then add 100 μL beads suspension to each tube    (beads volume/sample volume=1). Mix the 200 μL mixture by pipetting    up and down. Leave at room temperature.-   3. Incubate at room temperature for 10 minutes.-   4. Transfer the tubes to a magnet and let stand for at least 10    minutes for a complete separation of beads. Wait longer if    necessary. Then discard all 200 μL liquid phase. Make sure that    beads are not dispersing to avoid beads loss. Do not remove tubes    from the magnet yet.-   5. Add 200 μL freshly prepared 70% ethanol with the tubes still on    magnet, let stand for 30 sec, then discard the all liquid phase    using a pipette. Repeat one more time for a total of two ethanol    washes. For the second wash, remove as much liquid phase as    possible; the leftover liquid may affect results. Do not remove    tubes from the magnet yet.-   6. Air dry the beads in the tubes for around 10 minutes on magnet.    Make sure beads are completely dried and avoid over dry. It is    important to have all residual ethanol removed. Time needed to dry    may varies depend on the experimental environment. Check every 2    minutes to avoid over dry. Remove tubes from the magnet.-   7. Add 30 μL the “DNA Resuspension Buffer” that is already    equilibrate to room temperature to the dried beads. Flick the tubes    to suspend the beads, vortex, and pulse spin down if necessary.-   8. Transfer the tubes to a magnet and let stand for at least 5    minutes for a complete separation of beads. Wait longer if    necessary. Then transfer all 30 μL liquid phase to a set of new    tubes (or plates). Proceed to the next step of “First Stage Library    Amplification” immediately or store the mixture at −20° C.

i. First Stage Library Amplification (18 Rounds)

-   1. Pre-warm thermal cycler to run Program 12 (70° C. for 10 min, 18    cycles of: 94° C. for 30 sec+60° C. for 30 sec+72° C. for 1 min,    72° C. for 5 min, hold at 10° C.). Take “Amplification Buffer Mix    (buffer for Taq DNA Polymerase)” and “Amplification Primer Mix I    (primers base-pairing with adaptor for amplification, with the    ‘reverse primer’ consist of dUTP instead of thymine)” out from    −20° C. storage, thaw under room temperature, vortex to mix, spin    down, and keep at room temperature. Take “Amplification Enzyme Mix    (Taq DNA Polymerase)” out from −20° C. storage, flick gently to mix,    spin down, and keep on ice. Take “AMPure beads” out from 4° C.    storage and “DNA Resuspension Buffer” out from −20° C. storage,    equilibrate and thaw at room temperature for at least 30 minutes    before the next step of “First Round Library Purification.”-   2. Prepare a first stage amplification cocktail by mixing the    following components (the volume for each component is for one    amplification reaction): Amplification Buffer Mix: 10 μL,    Amplification Primer Mix I: 9.5 μL, and Amplification Enzyme Mix:    0.5 μL. Mix thoroughly by pipetting up and down at least 10 times    with 70% of total volume. Then spin down and put on ice. Use    immediately.-   3. Add 20 μL of the first stage amplification cocktail to each    sample tube from step (g)(8) for a total volume of 50 μL. Mix    thoroughly by pipetting up and down for at least 10 times. Spin down    and place on ice.-   4. Place the tube in the pre-warmed thermal cycler and start Program    12 (70° C. for 10 min, 18 cycles of: 94° C. for 30 sec+60° C. for 30    sec+72° C. for 1 min, 72° C. for 5 min, hold at 10° C.). Remove the    tube out of the thermal cycler, spin down the condensation and place    on ice. Proceed to the next step of “First Round Library    Purification” immediately or store the mixture at −20° C.

j. First Round Library Purification (Conducted at Room Temperature)

-   1. Make sure that “AMPure Beads” and “DNA Resuspension Buffer” are    thawed completely and are equilibrated to room temperature. This    usually takes at least 30 minutes. Vortex “AMPure Beads” and “DNA    Resuspension Buffer” to resuspend and mix. Leave the reagents at    room temperature. Do not spin down beads. Make sure “AMPure Beads”    are fully suspended before use. Prepare 70% ethanol freshly.-   2. Add 40 μL beads suspension to each tube from step (i)(4) for a    total volume of 90 μL (beads volume/sample volume=0.8). Mix the 90    μL mixture by pipetting up and down. Leave at room temperature.-   3. Incubate at room temperature for 10 minutes.-   4. Transfer the tubes to a magnet and let stand for at least 5    minutes for a complete separation of beads. Wait longer if    necessary. Then discard all 90 μL liquid phase. Make sure that beads    are not dispersing to avoid beads loss. Do not remove tubes from the    magnet yet.-   5. Add 200 μL freshly prepared 70% ethanol with the tubes still on    magnet, let stand for 30 sec, then discard the all liquid phase    using pipettes. Repeat one more time for a total of two washes. For    the second wash, remove as much liquid phase as possible; the    leftover liquid may affect results. Do not remove tubes from the    magnet yet.-   6. Air dry the beads in the tubes for around 10 minutes on magnet.    Make sure beads are completely dried, but avoid over drying. It is    important to have all residual ethanol removed. Time needed to dry    may varies depend on the experimental environment. Check every 2    minutes to avoid over drying. Remove tubes from the magnet.-   7. Add 50 μL “DNA Resuspension Buffer” that is already equilibrate    to room temperature to dried beads. Flick the tubes to suspend the    beads, vortex, and pulse spin down if necessary.-   8. Transfer the tubes to a magnet and let stand for at least 5    minutes for a complete separation of beads. Wait longer if    necessary. Then transfer all 50 μL liquid phase to a set of new    tubes (or plates). Leave at room temperature.-   9. At room temperature, add 40 μL beads suspension to each tube from    step (j)(8) for a total volume of 90 μL (beads volume/sample    volume=0.8). Mix the 90 μL mixture by pipetting up and down. Leave    at room temperature.-   10. Incubate at room temperature for 10 minutes.-   11. Transfer the tubes to a magnet and let stand for at least 5    minutes for a complete separation of beads. Wait longer if    necessary. Then discard all 90 μL liquid phase. Make sure that beads    are not dispersing to avoid beads loss. Do not remove tubes from the    magnet yet.-   12. Add 200 μL freshly prepared 70% ethanol with the tubes still on    magnet, let stand for 30 sec, then discard all liquid phase using    pipettes. Repeat one more time for a total of two ethanol washes.    For the second wash, remove as much liquid phase as possible; the    leftover liquid may affect results. Do not remove tubes from the    magnet yet.-   13. Air dry the beads in the tubes for around 10 minutes on magnet.    Make sure beads are completely dried and avoid over drying. It is    important to have all residual ethanol removed. Time needed to dry    may varies depend on the experimental environment. Check every 2    minutes to avoid over drying. Remove tubes from the magnet.-   14. Add 25 μL “DNA Resuspension Buffer” that is already equilibrate    to room temperature to dried beads. Flick the tubes to suspend the    beads, vortex, and pulse spin down if necessary.-   15. Transfer the tubes to a magnet and let stand for at least 5    minutes for a complete separation of beads. Wait longer if    necessary. Then transfer all 25 μL liquid phase to a set of new    tubes (or plates). Proceed to the next step of “Check Point I—cDNA    Quantification” immediately or store the mixture at −20° C.

k. Check Point I—cDNA Quantification

-   1. Quantify cDNA in library with double-strand DNA quantification    kit A. Leave at least 8.5 μL of sample for the next step of “rRNA    Depletion.” cDNA concentration in the sample should be greater than    10 pg/μL.

1. rRNA Depletion and Strand Selection

-   1. Pre-warm thermal cycler to run Program 13 (37° C.—10 min, 95°    C.—2 min, 50° C.—1 min, 65° C.—10 min, hold at 4° C.). Take    “Amplification Buffer Mix”, “rRNA Depletion Primer Mix (primer that    base-pair with specific human rRNA region)” and “Nuclease-free    Water” out from −20° C. storage, thaw at room temperature, vortex to    mix, spin down, and keep on ice. Take “Strand Selection Enzyme Mix    (uracil DNA glycosylase to degrade the specific cDNA strand    containing dUTP)”, “Amplification Enzyme Mix (Taq DNA Polymerases)”    and “Adaptor Cleavage Enzyme Mix (Restriction Endonuclease)” out    from −20° C. storage, flick gently to mix, spin down, and keep on    ice.-   2. Load a set of new PCR tubes/plates with 10 ng or at most 8.5 μL    of cDNA library. Add “Nuclease-free Water” to make a total volume of    8.5 μL if necessary.-   3. Prepare a strand selection cocktail by mixing the following    components (the volume for each component is for one strand    selection reaction): Amplification Buffer Mix: 5 μL, rRNA Depletion    Primer Mix: 10 μL, Strand Selection Enzyme Mix: 0.5 μL, and    Amplification Enzyme Mix: 1 μL. Mix thoroughly by pipetting up and    down at least 10 times with 70% of total volume. Then spin down and    put on ice. Use immediately.-   4. Add 16.5 μL of the strand selection cocktail to each sample tube    for a total volume of 25 μL. Mix thoroughly by pipetting up and down    for at least 10 times. Spin down and place on ice.-   5. Place the tube in the pre-warmed thermal cycler and start Program    13 (37° C. for 10 min, 95° C. for 2 min, 50° C. for 1 min, 65° C.    for 10 min, hold at 4° C.). Remove the tube out of the thermal    cycler, spin down the condensation, and place on ice.-   6. Pre-warm thermal cycler to run Program 14 (55° C. for 30 min,    95° C. for 5 min, hold at 4° C.).-   7. Prepare an adaptor cleavage cocktail by mixing the following    components (the volume for each component is for one adaptor    cleavage reaction): Amplification Buffer Mix: 5 μL, Adaptor Cleavage    Enzyme Mix: 2.5 μL, and Nuclease-free Water: 17.5 μL. Mix thoroughly    by pipetting up and down at least 10 times with 70% of total volume.    Then spin down and put on ice. Use immediately.-   8. Add 25 μL of the adaptor cleavage cocktail to each sample tube    from step (1)(5) for a total volume of 50 μL. Mix thoroughly by    pipetting up and down for at least 10 times. Spin down and place on    ice.-   9. Place the tube in the pre-warmed thermal cycler and start Program    14 (55° C. for 30 min, 95° C. for 5 min, hold at 4° C.). Remove the    tube out of the thermal cycler, spin down the condensation, and    place on ice. Proceed to the next step of “Second Stage Library    Amplification” immediately or store the mixture at −20° C.

m. Second Stage Library Amplification (8 Rounds)

-   1. Pre-warm thermal cycler to run Program 15 (95° C. for 2 min, 2    cycles of: 95° C. for 30 sec+60° C. for 90 sec, 6 cycles of: 95° C.    for 30 sec+65° C. for 90 sec, 65° C. for 5 min, hold at 4° C.). Take    “Amplification Buffer Mix” and “Amplification Primer Mix II (primers    base-pairing with adaptor for amplification)” out from −20° C.    storage, thaw under room temperature, vortex to mix, spin down, and    keep at room temperature. Take “Amplification Enzyme Mix” out from    −20° C. storage, flick gently to mix, spin down and keep on ice.    Take “AMPure beads” out from 4° C. storage and “DNA Resuspension    Buffer” out from −20° C. storage, equilibrate and thaw at room    temperature for at least 30 minutes before the step of “Second Round    Library Purification.”-   2. Prepare a second stage amplification cocktail by mixing the    following components (the volume for each component is for one    amplification reaction): Amplification Buffer Mix: 10 μL,    Amplification Primer Mix II: 39.5 μL, and Amplification Enzyme Mix:    0.5 μL. Mix thoroughly by pipetting up and down at least 10 times    with 70% of total volume. Then spin down and put on ice. Use    immediately.-   3. Add 50 μL of the second stage amplification cocktail to each    sample tube from step (1)(9) for a total volume of 100 μL. Mix    thoroughly by pipetting up and down for at least 10 times. Spin down    and place on ice.-   4. Place the tube in the pre-warmed thermal cycler and start Program    15 (95° C. for 2 min, 2 cycles of: 95° C. for 30 sec+60° C. for 90    sec, 6 cycles of: 95° C. for 30 sec+65° C. for 90 sec, 65° C. for 5    min, hold at 4° C.). Remove the tube out of the thermal cycler, spin    down the condensation, and place on ice. Proceed to the next step of    “Second Round Library Purification” immediately or store the mixture    at −20° C.

n. Second Round Library Purification (Conducted at Room Temperature)

-   1. Make sure that “AMPure Beads” and “DNA Resuspension Buffer” are    thawed completely and are equilibrated to room temperature. This    usually takes at least 30 minutes. Vortex “AMPure Beads” and “DNA    Resuspension Buffer” to resuspend and mix. Leave the reagents at    room temperature. Do not spin down beads. Make sure “AMPure Beads”    are fully suspended before use. Prepare 70% ethanol freshly.-   2. Add 100 μL beads suspension to each tube from step (m)(4) for a    total volume of 200 μL (beads volume/sample volume=1). Mix the 200    μL mixture by pipetting up and down. Leave at room temperature.-   3. Incubate at room temperature for 10 minutes.-   4. Transfer the tubes to a magnet and let stand for at least 10    minutes for a complete separation of beads. Wait longer if    necessary. Then discard all 200 μL liquid phase. Make sure that    beads are not dispersing to avoid beads loss. Do not remove tubes    from the magnet yet.-   5. Add 200 μL freshly prepared 70% ethanol with the tubes still on    magnet, stand for 30 sec, then discard the all liquid phase using    pipettes. Repeat one more time for a total of two ethanol washes.    For the second wash, remove as much liquid phase as possible; the    leftover liquid may affect results. Do not remove tubes from the    magnet yet.-   6. Air dry the beads in the tubes for around 10 minutes on magnet.    Make sure beads are completely dried and avoid over drying. It is    important to have all residual ethanol removed. Time needed to dry    may varies depend on the experimental environment. Check every 2    minutes to avoid over drying. Remove tubes from the magnet.-   7. Add 50 μL “DNA Resuspension Buffer” that is already equilibrate    to room temperature to dried beads. Flick the tubes to suspend the    beads, vortex, and pulse spin down if necessary.-   8. Transfer the tubes to a magnet and let stand for at least 5    minutes for a complete separation of beads. Wait longer if    necessary. Then transfer all 50 μL liquid phase to a set of new    tubes (or plates). Leave at room temperature.-   9. At room temperature, add 50 μL beads suspension to each tube from    step (n)(8) for a volume of 90 μL (beads volume/sample volume=1).    Mix the 100 μL mixture by pipetting up and down. Leave at room    temperature.-   10. Incubate at room temperature for 10 minutes.-   11. Transfer the tubes to a magnet and let stand for at least 5    minutes for a complete separation of beads. Wait longer if    necessary. Then discard all 100 μL liquid phase. Make sure that    beads are not dispersing to avoid beads loss. Do not remove tubes    from the magnet yet.-   12. Add 200 μL freshly prepared 70% ethanol with the tubes still on    magnet, stand for 30 sec, then discard all liquid phase using    pipettes. Repeat one more time for a total of two ethanol washes.    For the second wash, remove as much liquid phase as possible; the    leftover liquid may affect results. Do not remove tubes from the    magnet yet.-   13. Air dry the beads in the tubes for around 10 minutes on magnet.    Make sure beads are completely dried to avoid over drying. It is    important to have all residual ethanol removed. Time needed to dry    may varies depend on the experimental environment. Check every 2    minutes to avoid over drying. Remove tubes from the magnet.-   14. Add 25 μL “DNA Resuspension Buffer” that is already equilibrate    to room temperature to dried beads. Flick the tubes to suspend the    beads, vortex, and pulse spin down if necessary.-   15. Transfer the tubes to a magnet and let stand for at least 5    minutes for a complete separation of beads. Wait longer if    necessary. Then transfer all 25 μL liquid phase to a set of new    tubes (or plates). Proceed to the next step of “Check Point II—cDNA    Quantity and Quality” immediately or store the mixture at −20° C.

o. Check Point II—cDNA Quantity and Quality

-   1. Quantify cDNA in library with double-strand DNA quantification    kit A. cDNA Concentration should be greater than 1 nM.-   2. Check the quality of cDNA in library with double-strand DNA gel    electrophoresis array kit B. Most of the cDNA molecules should have    sizes round 300 base pair. The range of cDNA molecules should be    around 200 to 600 base-pair.

Example 3 Observed Gene Complexity and Tissue Specific Genes OverallDiversity of Detected Genes in Serum

We used three serums from three different individuals, indicated byserum A, B, and C, to investigate the number of different categories ofgenes that can be detected with the present method. By applying thepresent method, after sequencing and mapping genes having ENSEMBL geneannotation HG38, a large diversity of genes was observed with highconsistency over all three serums, with the “effective” serum inputvolume being 45 μL, 61 μL, and 61 μL, respectively, for serum A, B, andC. The yields of the sequencing reads were 40.4 million, 64.9 million,and 50.2 million, respectively. The number of genes detected from serumA, B, and C were 50606, 50949, and 50544, respectively, each of whichfell in the same 42 out of 44 human gene categories. These categoriesincluded, most importantly, protein-coding genes, lincRNA, miRNA, andsnRNA, etc., which were crucial for basic functions and regulations.With sequencing data from all libraries from each serum combined, thepercentages of genes detected in different categories annotated withENSEMBL gene annotation HG38 were as follows: in serum A: 95% of allprotein coding genes, 92% of all lincRNA, 41% of all miRNA, and 67% ofall snRNA were detected; in serum B: 95% of all protein coding genes,93% of all lincRNA, 42% of all miRNA, and 65% of all snRNA weredetected; in serum C: 94% of all protein coding genes, 93% of alllincRNA, 41% of all miRNA, and 63% of all snRNA were detected. Table 2below lists the specific numbers of genes detected in each of thefollowing gene categories: protein-coding gene, lincRNA, miRNA, andsnRNA (ENSEMBL gene annotation HG38 was used as reference genome). Thetotal number of genes for each category is shown in the parenthesisafter the name of each gene category.

TABLE 2 Protein-coding lincRNA Gene (19826) (7668) miRNA (4198) snRNA(1905) Num- Per- Num- Per- Num- Per- Num- Per- Serum ber cent ber centber cent ber cent A 18769 95% 7073 92% 1708 41% 1268 67% B 18784 95%7116 93% 1781 42% 1236 65% C 18716 94% 7096 93% 1718 41% 1197 63%

In summary, large and consistent gene diversity was observed among allthree serums with the present method. Genes were detected in the same 42out of all 44 human gene categories with all three serums covering bothrecurrence and non-recurrence status. Important categories such asprotein coding genes, lincRNA, miRNA, and snRNA were studied asexamples. Almost all protein coding genes (>94%), most of lincRNA(>92%), and around 41% and 65% of all miRNA and snRNA, respectively,could be detected using about 45 μL of serum A and about 61 μL of eachof serum B and serum C. High consistency of gene diversity was observedamong three serums, even though they were from different individuals.

Overall Diversity of Detected Genes in Cell-Free Saliva

Cell-free saliva D from an individual was analyzed to investigate thediversity of genes that could be detected. With the current methodapplied, after sequencing and mapping harvested data to ENSEMBL geneannotation HG38, a large diversity of genes was observed. From the“effective” volume of 13 μL cell-free saliva input, around 6.4 millionsequencing reads were generated and a total of 24897 genes was detected.These genes belong to various of categories, and most importantly,protein-coding genes, lincRNA, miRNA, snRNA, etc. 14215 out of all 19826protein-coding genes, 3040 out of all 7668 lincRNA, 211 out of all 4198miRNA, and 179 out of all 1905 snRNA were identified. Table 3 belowlists the specific numbers of genes detected in each of the followinggene categories: protein-coding gene, lincRNA, miRNA, and snRNA (ENSEMBLgene annotation HG38 was used as reference genome), and thecorresponding percentage. The total number of genes for each category isshown in the parenthesis after the name of each gene category.

TABLE 3 Protein-coding lincRNA Cell- Gene (19826) (7668) miRNA (4198)snRNA (1905) free Num- Per- Num- Per- Num- Per- Num- Per- Saliva bercent ber cent ber cent ber cent D 14215 72% 3040 40% 211 5% 179 9%

In summary, a large diversity of genes was detected from cell-freesaliva with the present method. There were 24897 different genesdetected from 13 μL cell-free saliva and the genes were also diverse interms of categories. Using the four important gene categories asexamples, the majority (72%) of protein-coding genes, a large proportion(40%) of lincRNA as well as some miRNA, and snRNA were identified.

Overall Diversity of Detected Genes in IVF Culture Media

The diversity of genes that could be detected in IVF culture media wasstudied using exemplary IVF culture media E. With the method above,after sequencing and mapping harvested data to ENSEMBL gene annotationHG38, numerous genes from a diverse of gene categories were observed.Around 5.2 million sequencing reads were generated and as many as 31346genes from various gene categories were detected in 7 μL IVF culturemedia input, with the most important gene categories beingprotein-coding genes, lincRNA, miRNA, snRNA, etc. Among allprotein-coding genes, 15163 could be identified; the numbers were 4431for lincRNA, 420 for miRNA, and 483 for snRNA. Table 4 below lists thespecific numbers of genes detected in each of the following genecategories: protein-coding gene, lincRNA, miRNA, and snRNA (ENSEMBL geneannotation HG38 was used as reference genome), and the correspondingpercentage. The total number of genes for each category is shown in theparenthesis after the name of each gene category.

TABLE 4 Protein-coding lincRNA Gene (19826) (7668) miRNA (4198) snRNA(1905) IVF Num- Per- Num- Per- Num- Per- Num- Per- Media ber cent bercent ber cent ber cent E 15163 76% 4431 58% 420 10% 483 25%

In summary, a large number of genes from a variety of categories wasidentified in IVF culture media with the present method. A total of31346 genes were detected in 7 μL IVF culture media and these genes werefrom diverse gene categories. For example, among the most important genecategories, the majority (76%) of protein-coding genes, a largeproportion (58%) of lincRNA, as well as a proportion of miRNA (10%) andsnRNA (25%) were identified.

Discovery of Tissue-Specific Genes in Serum

The present method also allows the detection of tissue specific genes,especially those genes specific to tissues that are difficult or evenimpossible to sample using biopsy. The present method for sequencingusing serum provides an advantage over traditional sequencing usingbiopsy samples. Three tissues were selected as examples where biopsysampling is difficult or impossible: brain, bone marrow, and peripheralnervous system (PNS). The present method was applied using three serumsfrom three different individuals: serum A, B, and C (same as the serumsdescribed above). RNA sequencing was conducted as described in Example 2and the data was then mapped to ENSEMBL gene annotation HG38 and matchedusing TiGER tool (Tissue-specific Gene Expression and Regulation,Bioinformatics Lab at Wilmer Institute, Johns Hopkins University) whichprovides tissue-specific gene expression profile for genes specific tocertain tissues.

With the present method, when “effective” serum input volume for eachserum was used (45 μL for serum A, 61 μL for serum B, and 61 μL forserum C), 176 brain specific genes, 192 bone marrow specific genes, and78 PNS specific genes were detected in serum A; 175 brain specificgenes, 191 bone marrow specific genes, and 78 PNS specific genes weredetected in serum B; and 176 brain specific genes, 189 bone marrowspecific genes, and 78 PNS specific genes were detected in serum C(Table 5). The total numbers of tissue specific genes for the threetissues/organs matched using TiGER tool are shown in the first row.

TABLE 5 Brain Specific Bone Marrow Specific PNS Specific AU in Data Base176 192 78 Serum A 176 191 78 Serum B 175 191 78 Serum C 176 189 78

FIGS. 2A-2I further show that the TPM (transcripts per million) of genesspecific to these three tissues were mostly larger than 4. Success indetecting specific genes for important tissues and organs reveals thepotential of applying the present method to serums as an alternative totraditional in-tissue sampling during diagnosis and prognosis ofdiseases related to those specific tissues or organs.

Exemplary Embodiments

Exemplary embodiments provided in accordance with the presentlydisclosed subject matter include, but are not limited to, the claims andthe following embodiments:

1. A method for detecting a plurality of ribonucleic acids (RNAs) in abiological sample from a subject, the method comprising:

(a) constructing a cDNA library from the plurality of RNAs in thebiological sample, wherein an effective volume of the biological sampleused to construct the cDNA library is less than or equal to about 1 mL;and

(b) detecting RNA genome equivalents in the cDNA library.

2. The method of embodiment 1, wherein the effective volume of thebiological sample is less than or equal to about 500 μL.

3. The method of embodiment 1 or 2, wherein the effective volume of thebiological sample is less than or equal to 250 μL.

4. The method of embodiment 1 or 2, wherein the effective volume of thebiological sample is less than or equal to about 100 μL.

5. The method of any one of embodiments 1 to 4, wherein the biologicalsample is a cell-free biological sample.

6. The method of any one of embodiments 1 to 5, wherein at least about100 different genes are detected.

7. The method of any one of embodiments 1 to 6, wherein at least about1000 different genes are detected.

8. The method of any one of embodiments 1 to 7, wherein at least about10,000 different genes are detected.

9. The method of any one of embodiments 1 to 8, wherein at least about30,000 different genes are detected.

10. The method of any one of embodiments 1 to 9, wherein the differentgenes comprise different categories of RNAs.

11. The method of embodiment 10, wherein the different categories ofRNAs are selected from the group consisting of mRNA, lincRNA, miRNA,snRNA, and combinations thereof

12. The method of any one of embodiments 1 to 11, wherein the biologicalsample is a whole blood sample, a plasma sample, a serum sample, asaliva sample, a cell culture media sample, a urine sample, an amnioticfluid sample, a mucus sample, a semen sample, a vaginal fluid sample, asputum sample, a cerebrospinal fluid sample, a lymphatic fluid sample,an ocular fluid sample, a sweat sample, or a stool sample.

13. The method of embodiment 12, wherein the biological sample is aserum sample, a plasma, a saliva sample, or a cell culture media sample.

14. The method of embodiment 13, wherein the cell culture media sampleis a serum sample or a plasma sample.

15. The method of embodiment 13, wherein the cell culture media sampleis an in vitro fertilization (IVF) culture media sample.

16. The method of any one of embodiments 1 to 15, wherein step (a)comprises:

(a1) breaking up lipid bilayers and/or ribonucleoprotein complexes inthe biological sample;

(a2) removing deoxyribonucleic acids (DNAs) in the biological sample;

(a3) synthesizing a plurality of short, double-stranded complementaryDNAs (cDNAs) oligonucleotides using the RNAs in the biological sample astemplates;

(a4) ligating at least one adaptor to an end of each short,double-stranded cDNA oligonucleotide to generate a plurality ofadaptor-ligated, double-stranded cDNA oligonucleotides;

(a5) amplifying the plurality of adaptor-ligated, double-stranded cDNAoligonucleotides using primers that hybridize to the adaptor;

(a6) selecting one strand of each adaptor-ligated, double-stranded cDNAoligonucleotide; and

(a7) amplifying the selected strand of each adaptor-ligated,double-stranded cDNA oligonucleotide to generate the cDNA library,wherein the cDNA library comprises cDNA oligonucleotides having the samesequences as the sequences of the genomic DNAs from which the RNAs aretranscribed.

17. The method of embodiment 16, wherein step (a4) comprises ligatingtwo adaptors each to one end of each short, double-stranded cDNAoligonucleotide to generate a plurality of adaptor-ligated,double-stranded cDNA oligonucleotides.

18. The method of embodiment 17, wherein the two adaptors are the same.

19. The method of embodiment 17, wherein the two adaptors are different.

20. The method of any one of embodiments 16 to 19, wherein step (a3)comprises:

(a3.1) synthesizing a plurality of first strand cDNAs by reversetranscribing the RNAs in the biological sample;

(a3.2) fragmenting the plurality of first strand cDNAs to generate aplurality of cDNA fragments;

(a3.3) ligating a 3′ primer to the 3′ end of each cDNA fragment in theplurality of cDNA fragments; and

(a3.4) synthesizing the plurality of short, double-stranded cDNAoligonucleotides using a targeting primer and the plurality of cDNAfragments as templates, wherein the targeting primer comprises a firstportion comprising a sequence complementary to the sequence of the 3′primer and a second portion comprising a degradable sequence.

21. The method of embodiment 20, wherein the degradable sequencecomprises a uracil DNA glycosylase recognition sequence.

22. The method of embodiment 20, wherein the 3′ primer is anoligonucleotide comprising identical nucleotides.

23. The method of embodiment 22, wherein the 3′ primer is a poly(G)oligonucleotide, a poly(C) oligonucleotide, a poly(A) oligonucleotide, apoly(T) oligonucleotide, or a poly(U) oligonucleotide.

24. The method of embodiment 23, wherein the 3′ primer is a poly(G)oligonucleotide.

25. The method of embodiment 20 or 24, wherein the sequence of the firstportion is a poly(C) oligonucleotide.

26. The method of any one of embodiments 16 to 25, further comprisingremoving the RNAs in the biological sample.

27. The method of embodiment 26, wherein the RNAs are removed using ametal ion and/or heat shock.

28. The method of any one of embodiments 17 to 27, wherein one of theadaptors comprises a degradable sequence.

29. The method of embodiment 28, wherein the degradable sequencecomprises a uracil DNA glycosylase recognition sequence.

30. The method of any one of embodiments 16 to 29, wherein the pluralityof adaptor-ligated, double-stranded cDNA oligonucleotides is purifiedusing a charge-based purification method prior to step (a5).

31. The method of embodiment 30, wherein the charge-based purificationmethod comprises using beads.

32. The method of any one of embodiments 28 to 31, wherein in step (a6),the strand comprising the adaptor that comprises the degradable sequenceis degraded.

33. The method of embodiment 32, wherein the strand is degraded by auracil DNA glycosylase.

34. The method of any one of embodiments 16 to 33, further comprisingremoving cDNA oligonucleotides that encode ribosomal RNAs (rRNAs) afterstep (a6) and prior to step (a7).

35. The method of embodiment 34, wherein an rRNA primer is used totarget the cDNA oligonucleotides encoding rRNAs.

36. The method of any one of embodiments 1 to 35, wherein step (b)comprises:

(b1) sequencing the cDNA library to detect the RNA genome equivalents.

37. The method of embodiment 36, wherein at least 10,000 sequencingreads are obtained.

38. The method of embodiment 37, wherein at least 50,000 sequencingreads are obtained.

39. The method of embodiment 38, wherein at least 1 million sequencingreads are obtained.

40. The method of any one of embodiments 36 to 39, wherein step (b)further comprises:

(b2) mapping the RNA genome equivalents detected to different categoriesof RNAs.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by thoseskilled in the relevant arts, once they have been made familiar withthis disclosure, that various changes in form and detail can be madewithout departing from the true scope of the invention in the appendedclaims. The invention is therefore not to be limited to the exactcomponents or details of methodology or construction set forth above.Except to the extent necessary or inherent in the processes themselves,no particular order to steps or stages of methods or processes describedin this disclosure, including the Figures, is intended or implied. Inmany cases the order of process steps may be varied without changing thepurpose, effect, or import of the methods described.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents (patents,published patent applications, and unpublished patent applications) isnot intended as an admission that any such document is pertinent priorart, nor does it constitute any admission as to the contents or date ofthe same.

1. A method for detecting a plurality of ribonucleic acids (RNAs) in abiological sample from a subject, the method comprising: (a)constructing a cDNA library from the plurality of RNAs in the biologicalsample, wherein an effective volume of the biological sample used toconstruct the cDNA library is less than or equal to about 1 mL; and (b)detecting RNA genome equivalents in the cDNA library.
 2. The method ofclaim 1, wherein the effective volume of the biological sample is (a)less than or equal to about 500 μL; (b) less than or equal to 250 μL; or(c) less than or equal to about 100 μL.
 3. (canceled)
 4. (canceled). 5.The method of claim 1, wherein the biological sample is a cell-freebiological sample.
 6. The method of claim 1, wherein (a) at least about100 different genes are detected; (b) at least about 1000 differentgenes are detected; (c) at least about 10,000 different genes aredetected, or (d) at least about 30,000 different genes are detected.7.-9. (canceled)
 10. The method of claim 1, wherein the different genescomprise different categories of RNAs.
 11. The method of claim 10,wherein the different categories of RNAs are selected from the groupconsisting of mRNA, lincRNA, miRNA, snRNA, and combinations thereof. 12.The method of claim 1, wherein the biological sample is a whole bloodsample, a plasma sample, a serum sample, a saliva sample, a cell culturemedia sample, a urine sample, an amniotic fluid sample, a mucus sample,a semen sample, a vaginal fluid sample, a sputum sample, a cerebrospinalfluid sample, a lymphatic fluid sample, an ocular fluid sample, a sweatsample, or a stool sample.
 13. (canceled)
 14. The method of claim 12,wherein the cell culture media sample is a serum sample or a plasmasample or an in vitro fertilization (IVF) culture media sample. 15.(canceled)
 16. The method of claim 1, wherein step (a) comprises: (a1)breaking up lipid bilayers and/or ribonucleoprotein complexes in thebiological sample; (a2) removing deoxyribonucleic acids (DNAs) in thebiological sample; (a3) synthesizing a plurality of short,double-stranded complementary DNAs (cDNAs) oligonucleotides using theRNAs in the biological sample as templates; (a4) ligating at least oneadaptor to an end of each short, double-stranded cDNA oligonucleotide togenerate a plurality of adaptor-ligated, double-stranded cDNAoligonucleotides; (a5) amplifying the plurality of adaptor-ligated,double-stranded cDNA oligonucleotides using primers that hybridize tothe adaptor; (a6) selecting one strand of each adaptor-ligated,double-stranded cDNA oligonucleotide; and (a7) amplifying the selectedstrand of each adaptor-ligated, double-stranded cDNA oligonucleotide togenerate the cDNA library, wherein the cDNA library comprises cDNAoligonucleotides having the same sequences as the sequences of thegenomic DNAs from which the RNAs are transcribed.
 17. The method ofclaim 16, wherein step (a4) comprises ligating two adaptors each to oneend of each short, double-stranded cDNA oligonucleotide to generate aplurality of adaptor-ligated, double-stranded cDNA oligonucleotides,wherein the two adaptors are the same or different.
 18. (canceled) 19.(canceled)
 20. The method of claim 16, wherein step (a3) comprises:(a3.1) synthesizing a plurality of first strand cDNAs by reversetranscribing the RNAs in the biological sample; (a3.2) fragmenting theplurality of first strand cDNAs to generate a plurality of cDNAfragments; (a3.3) ligating a 3′ primer to the 3′ end of each cDNAfragment in the plurality of cDNA fragments; and (a3.4) synthesizing theplurality of short, double-stranded cDNA oligonucleotides using atargeting primer and the plurality of cDNA fragments as templates,wherein the targeting primer comprises a first portion comprising asequence complementary to the sequence of the 3′ primer and a secondportion comprising a degradable sequence.
 21. The method of claim 20,wherein fa) the degradable sequence comprises a uracil DNA glycosylaserecognition sequence; or (b) the 3′ primer is an oligonucleotidecomprising identical nucleotides; or (c) the sequence of the firstportion is a poly(C) oligonucleotide.
 22. (canceled)
 23. The method ofclaim 21, wherein the 3′ primer is a poly(G) oligonucleotide, a poly(C)oligonucleotide, a poly(A) oligonucleotide, a poly(T) oligonucleotide,or a poly(U) oligonucleotide.
 24. (canceled)
 25. (canceled)
 26. Themethod of claim 16, further comprising removing the RNAs in thebiological sample.
 27. The method of claim 26, wherein the RNAs areremoved using a metal ion and/or heat shock.
 28. The method of claim 17,wherein one of the adaptors comprises a degradable sequence.
 29. Themethod of claim 28, wherein the degradable sequence comprises a uracilDNA glycosylase recognition sequence.
 30. The method of claim 16,wherein the plurality of adaptor ligated, double-stranded cDNAoligonucleotides is purified using a charge-based purification methodprior to step (a5).
 31. The method of claim 30, wherein the charge-basedpurification method comprises using beads.
 32. The method of claim 28,wherein in step (a6), the strand comprising the adaptor that comprisesthe degradable sequence is degraded.
 33. The method of claim 32, whereinthe strand is degraded by a uracil DNA glycosylase.
 34. The method ofclaim 16, further comprising removing cDNA oligonucleotides that encoderibosomal RNAs (rRNAs) after step (a6) and prior to step (a7).
 35. Themethod of claim 34, wherein an rRNA primer is used to target the cDNAoligonucleotides encoding rRNAs.
 36. The method of claim 1, wherein step(b) comprises: (b1) sequencing the cDNA library to detect the RNA genomeequivalents.
 37. The method of claim 36, wherein (i) at least 10,000sequencing reads are obtained; (ii) at least 50,000 sequencing reads areobtained; or (iii) at least 1 million sequencing reads are obtained. 38.(canceled)
 39. (canceled)
 40. The method of claim 36, wherein step (b)further comprises: (b2) mapping the RNA genome equivalents detected todifferent categories of RNAs.